Investigations Into the Etiology of Dermatitis Herpetiformis

INVESTIGATIONS INTO THE ETIOLOGY OF DERMATITIS HERPETIFORMIS 1. THE EFFECTS OF SERUM FROM DERMATITIS HEEPETIFOEMIS CASES ON THE SPREADING PROPERTY OF HYALURONIDASE*

J. O'D. ALEXANDER, M.B., CH.B., F.R.F.P.S.(G)

The etiology of dermatitis herpetiformis is obscure although numerous theories have been put forward to explain its occurrence. Recently Szodoray (1) reported having examined sections

measurement is required and a dye, which can be easily seen without leaving permanent staining, is essential. A short series of pilot experiments in

animals was therefore made to determine the

of skin from active lesions stained for mucopoly- most suitable dye and the accuracy of surface saccharides. From his examinations he concluded measurements.

that there was evidence of increased hyaluron-

idase activity in the skin of these cases. His

MATERIALS AND METHODS

finding prompted an investigation into the role Animals. Male adult albino mice and young which this enzyme might play in the etiology of male albino rats were used. this disease. Hyaluronictase. 50 units of commercial hyaluHyaluronidase (the spreading factor of Duran- ronidase (Hyalase Benger) were injected for each Reynals) is an enzyme which is found principally

measurement.

Indicators. Three indicators were tested. 1) in the testis but considerable amounts arc also known to occur in the skin. Testicular hyaluron- India ink suspended in sterile saline in concenidase acts not only on the hyaluronic acid of the trations of 1 in 2, 1 inS, and 1 in 10. 2) Evans blue 0.24% dissolved in sterile saline 0.9% concentraskin but also on the polysaccharide of cornea and tion. 3) Vital New Red. India ink, because its cartilage. The exact substrate of the hyaluronidase sharp definition would give accurate measurenative to the skin has not been stated hut it may ments, was used to compare with the other two. consist of not only hyaluronic acid but also acid Vital New Red was tried because, being of a pink mucopolysaccharides present in the skin. The tint, it might subsequently be less noticeable on

fact that testicular hyaluronidase has more the skin of patients. Injections. A 1.0 ml tuberculin syringe graduo than one substrate suggests that it may be ated in 0.01 ml divisions was used with a gauge 2t hypodermic needle. Approximately 0.03 ml of test solution was injected intradermally, taking aboue 2 seconds to make the actual injections. The needlThe hyaluronidase in skin may likewise consist of was inserted bevel uppermost, parallel to the skin more than one enzyme. surface and just far enough to occlude the opening In the present paper the method chosen for of the needle. assessing hyaluronidase activity has been the Measurements. Two methods were used. 1) the

composed of more than one enzyme, some of which act by dcpolymerization and others by hydrolysis of the resultant depolymerized units.

measurement of the rate of skin spreading. dyed area was traced out on small cellophane Previous work on the action of the spreading fac-

tor in the skin was carried out by Opsahl (2) in animals, using India ink as an indicator. She made measurements by removing the anterior abdominal wall after a fixed period and mapping the area stained with dye on the under surface of the skin. The animals used were rats. Juhlin (3) made measurements on the ear of rabbits using Evans Blue and hemoglobin as indicators. Re

cards with a ball point pen. 2) The dyed areas were regarded as ellipses and the longest diameter and the one at right angles to this were measured. Timing of measurements. 1) At half hour intervals for hours and 2) at 5 minute intervals for 40 minutes.

Site of injections. The hair was removed by plucking two days previously to ensure the unifornuty of the metabolic phase of the skin in all animals. The areas chosen were 1) the anterior ab-

calculated his areas by regarding them all as dominal wall and 2) the flanks. ellipses. Opsahl's method is obviously not ap RESULTS OF PILOT EXPERIMENTS plicable to humans. A reasonably accurate surface * From the Department of Dermatology, Glas-

gow Royal Infirmary, Glasgow, Scotland. Received for publication August 13, 1960. 39

Indicators. India ink was unsuitable because of color and permanence. Vital New Red was also unsuitable because of the extreme difficulty in

40

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

detecting the margin of the dyed area. Even in rats, where the naked skin has a bluish tint, it was very difficult. Those measurements which were made, however, proved to be accurate in comparison with the measurements on the under surface of the abdominal skin. Nevertheless, the possihility of mistakes was too groat to allow its use in pink human skins. Evans Blue on the other hand proved very satisfactory. It was easy to soc and the margin was usually clear cut. Syringes and needles. These proved to be quite satisfactory. Amount of the injection. Difficulty was exper-

ienced in obtaining a wheal of uniform size. Theoretically such a wheal would be elicited every time by the injection of 0.03 ml but the thinness of the skin and the small size of the

however faint (which indicated the presence of

dye), was regarded as the edge of the wheal. Measurement of the under surface of the skin showed this to he correct and the method subsequently proved very satisfactory in patients. The area of the wheal was calculated from the formula A = erab where a and b are the respective radii of the ellipse. Timing of the measurements. After examination of the various intervals it was finally decided that measurements at intervals of 10 minutes for 40

minutes would cover the period of active spreading satisfactorily. HUMAN EXPERIMENTs

Materials and Methods Hyaluronidase. Approximately 25 units of com-

animal made it technically very difficult to ensure mercial enzyme was included in each injection injecting the same amount each time. Further- when enzyme was required. more even if a threaded shaft to the syringe Indicator. 0.24% Evans Blue in Normal saline. plunger had been used it would not have helped Injections. The syringe needle and technique greatly because the inj ections would still have to was the same as that used for the animal experibe given slowly and the slightest movement of ments. 0.03 ml was injected. (see figs 1 and 2.) Site of the injections. A preliminary trial showed the animal resulted in the needle coming out of

that there was no gross difference in the rate of spreading in the skin on any surface of the foreestimated visually. A well-made, all glass syringe arms, on the upper back or the buttocks. For conwith a closely fitting plunger is essential. venience the forearm was chosen and several Site of injection. There was no apparent dif- separate injections could be given in one testing ference in the rate of spread between the skin of period. the flanks and of the anterior abdominal wall. Measurements. At 10 minute intervals for 40 Measurements. Mapping out areas by tracing minutes, using the criteria for the limits of the was found to be impracticable and inaccurate. It wheals as outlined for animals. (see figs, 3 and 4.). Test substances injected: was decided to regard all areas as ellipses acControl A. Evans Blue 0.24% in saline. cording to Juhlin's method. This proved emiControl B. Control A plus 25 units of Hyaluroni-

the skin. The size of the wheal was therefore

nently satisfactory. The wheals were easily dase. measured, quickly and accurately. The furthermost point showing alteration of color of the skin,

The following substances were each tested by adding to controls A and B:

rfrflr

Fea. 1. Position of the Needle immediately prior to the injection

HYALURONIDASE IN DERMATITIS HERPETIFOEMIS

41

at "

FIG. 2. The injection completed showing the approximate size

0

itt ,;

,

FIG. 3. Showing a representative trio of injections immediately after they have been completed. The one on the right contains dye and saline only nnd that in the centre contains 25 units of hyaluronidase in addition. The one on the left contains dye plus serum from a case of dermatitis herpetiformis but no hyaluronidase. 1. 0.25 mg hydrocortisone acetate. 2. Serum from a case of senile dermatitis herpetiformis before and after treatment with prednisolone. 3. Serum from 3 cases of active adult dermatitis

herpetiformis and one after treatment with dapsone.

4. Serum from a normal person. Tests were also carried out using fluid from blisters from a case of adult dermatitis herpetiformis. from a case of senile dermatitis herpetiformis and from a case of burns.

The sera and blister fluids used in these tests were all passed through a Seitz filter mixed with an

Test subjects. The tests were mostly carried out

on patients suffering from dermatitis herpetiformis either in an active or controlled state, using

the unaffected skin. Normal volunteers without skin disease and volunteers with other skin disorders were also used. A statistical analysis of the results in the different groups of the test subjects

showed that the state of the subject used for the injection made no significant difference to the result. The tests in all the different subjects were, therefore, grouped together as a whole in assessing the results of the different injections.

equal volume of Evans Blue (0.24%) in normal

saline and retested for sterility before the in-

RESULTS

jections were made. They were stored in rubber capped bottles at 4°C between tests. If any were found to have become infected they were discarded.

persons. Before discussing the results it is necessary to explain how these have been determined.

952 test injections were made in about 50

42

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

-

'.4. kA-t FIG. 4. Showing the same trio of injections as in Fig. 3 half an hour later. The injection containing hyaluronidase (in the centre) has spread at a greater rate. The ink marks show the visible limits of the dyed areas. TABLE 1 Showing example of the method used to calculate the

mean rate of spread of dye for any given injection Time of Measurement

after Injection (in minutes)

O (t1) 10 (t5)

20 (ta) 30 (t4)

40 (ts)

Total =

Actual in Area Over the Area of Increase Initial Area at 0 Minutes Dyed Skin (sq. mm.) (sq. mm.)

0 (ai) 9.4 (as) 15.7 (a,)

28.3 37.7 44.0 56.6 56.6

28.3 (a4)

28.3 (a,)

against the actual area at 20 minutes. The resultant graph showed that these readiugs lay along a straight line (Fig. 5). Moreover, when the various graphic points were replotted as separate groups according to the size of the initial wheal the readings were still about a straight line, the lines in the different groups being more or less parallel (Figz. 6—9). This suggests that the method of comparing minute-column increases was reasonably accurate

and thnt the size of the initial wheal could be ignored.

The results were analyzed statistically using the 't' test. In every instance where a difference between rate of spread with and without the test substance is shown, this difference has been dem-

100

(t+t2+

t3+t4+t5)

81.7

(ai+as + a3+a4+ a5)

Mean rate of spread = 81 .7/100 = 0.82 sq. mm. per minute, Owing to the difficulties of producing nn exactly

onstrated to he statistically significant. For example in the case of test serum 1 (Table 2) the re-

sults in column A refer to a comparison of the spreading rate of dye plus serum on the one hand and of dye plus hyaluronidase on the other. Twelve pairs of injections (each pair done on the same test

subject and twelve test subjects injected) were compared statistically for the rate of spread and the results showed that the rate was significantly

similar sized wheal with each injection a suitable method of calculation is required to try to offset less with serum plus dye than with hyaluronidase these discrepancies. The factor to be measured is plus dye. Similarly in column C the results show the rate of spread. This is obtained by dividing the that when serum 1 is added to dye plus hyaluroniincrease in area in square mms. by the time in dase the rate of spread is accelerated. minutes. A single reading might give a widely varying answer and therefore the mean rate of The results indicated that spread is obtained as shown in Table 1. This would 1. The spreading effect of the various test sera

give an excellent menns of comparison of different used was less than the spreading effect of hyinjections but for one factor. Does the initial size aluronidase (Column A Table 2) although in the

of the wheal have any pronounced influence on the rate of spread? To test this, a series of readings were plotted graphically as follows. The increase between 0 and 30 minutes (a4—a,) and between 10 and 40 minutes (as—as) were summated and plotted

case of sera numbers 4 and 5 the difference was not significant.

2. Normal serum had an inhibitory effect on the rate of spread of dye alone but all the other

HYALURONIDASE IN DERMATITIS HERPETIFORMIS

-se' •

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FIG. 5. Graph showing that the readings plotted as indicated lie along a straight line

sera had no significant effect (Column B Table

There was no accelerating effect with normal

2).

serum. 5. Blister fluid from a case of adult dermatitis

3. Serum from cases of untreated dermatitis herpetiformis had an accelerating effect on the spreading property of hyaluronidase (Column C Table 2). In the case of serum number 5 this accelerating effect although pronounced was just insufficient to be significant (at P = 0.05). The effect was significant in the other sera (numbers 1, 3 and 4). 4. This accelerating effect was abolished after the appropriate treatment with either prednisolone or dapsone (numbers 2 and 5 respectively).

herpetiformis (number 8) and from a case of burns (number 10) had less spreading effect than had hyaluronidase (Table 3 Column A). The fluid from a blister of senile dermatitis herpetiformis

(number 9) had a spreading effect not significantly different from hyaluronidase.

6. Blister fluids numbers 8 and 10 had no significant influence on the rate of spread of dye

alone but fluid number 9 had an accelerating effiect (Table 3 Column B).

44

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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FIGs. 6—9. Graphs showing that when the readings in Fig. 5 are regrouped according to size of the initial wheal and plotted in a similar manner they still lie about a straight line and that the lines of the different graphs are more or less parallel. This suggests that the size of the initial wheal did not influence the rate of spread of the dye in this series of experiments.

7. Blister fluids numbers 8 and 10 had an ac9. The addition of hydrocortisone acetate to celerating effect on the spreading property of blister fluid from senile dermatitis herpetiformis hyaluronidase (Table 3 Column C) but fluid resulted in inhibition of the previous accelerating number 9 had no significant influence on the rate of spread of the dye plus hyaluronidase although actually accelerating it a little.

effect of this fluid on spreading rate of dye (Table 3 comparing numbers 9 and 12 in Column B) but

(Table 3 Column A number 1) and in fact had an inhibitory effect on the rate of spread of dye with or without the addition of hyaluronidase. (Table 3 Columns B and C).

C). Similarly this steroid inhibited the ac-

otherwise had no significant effects (Table 3 8. Hydrocortisone acetate had significantly less spreading effect than had hyaluronidase Comparing numbers 9 and 12 in Columns A and celerating effect of burn blister fluid on hyaluronidase spreading rate (Table 3 Column B numbers 10 and 13).

45

HYALURONIDASE IN DERMATITIS HERPETIFORMIS

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DISCUSSION

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of active dermatitis herpetiformis, however,

The action of hyaluronidase in increasing the present a different and very interesting picture. In these cases it has been shown that such serum rate of spread of diffusion of substances in solunot only does not inhibit the spreading action of tion or finely divided suspension through the hyaluronidase but actually accelerates it. The tissues is known to be inhibited by several suboverall extent of spread is not materially affected stances, amongst which are normal human serum, as was shown by measurement of the dyed areas A.C.T.H. and steroids. In the experiments described above this has been confirmed for normal after 24 hours had elapsed since the injections. human serum and for hydrocortisone acetate. The There was no significant difference in the size of results of the tests using various blister fluids are these areas whether or not hyaluronidase was used confusillg and inconsistent and do not permit of in the injection and whether or not serum was any conclusions being drawn. This is partially due p esent as well. The rate of spread over the first to the fact that the contents of blister fluids are 40 minutes after the injection, however, was significantly increased if serum from active cases very variable in themselves. The results of the injections of serum from cases of dermatitis herpetiformis was incorporated in

46

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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the injection along with hyaluronidase. Moreover, serum from two of these patients, after

given to the patient from whom the serum is taken. Dapsone given to the patients on whom

their disease had been controlled, on the one hand

the tests were made did not have any noticeable

with prednisolone and in the other case with effect. That there is actually some additional facdapsone, did not show this accelerating effect and

behaved in the same manner as normal serum. Heating one of the active sera to 56°C for 2 hours did not alter its accelerating effect.

These results suggest that there is present in serum from active dermatitis herpetiformis a

tor present in the serum of these patients is suggested by the fact that the spreading action of hyaluronidase is accelerated. If it were that there was merely an absence of the usual inhibitory power of normal serum an accelerating effect would not have been observed.

way that this factor is heat stable. Its effect is

It is as yet a matter of conjecture as to how prednisolone and dapsone exert their effect on this factor. It does not appear to be through the

inhibited by both prednisolone and dapsone when

medium of the adrenal glands since in this case it

factor which accelerates the action of hyaluronidase. It appears from the one serum tested in this

47

HYALTJRONIDASE IN DERMATITIS IIERPETIFORMIS

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would be expected that with either of these treatments there would be a fall of circulating eosino-

subj ect who was being treated with prednisolone

phils. The latter is an index of adrenal activity and it has been shown that with dapsone therapy

Furthermore three other test subjects were tested before and after receiving 40 units of

gave results similar to the other test subjects.

this is not the case (Alexander (4)). These results A.C.T.H. and there was no significant change in do not therefore furnish evidence that control of the results. Whilst it has therefore not been con-

dermatitis herpetiformis is necessarily through the medium of the adrenal glands. It is possible

clusively proved that these drugs achieve their effect by inhibiting hyaluronidase directly it

that the amount of either prednisolone or dapsone

seems unlikely that this is the mechanism of their

present in the serum might actually inhibit the

action. Theie is, however, as the results show,

effect of the hyaluronidase directly as was shown

some additional factor present in dermatitis

to be the case with hydrocortisone acetate. The herpetiformis, which is inhibited by these drugs amount of dapsone present in the test subjects did (and possibly by sulfapyridine, which has a not appear to influence the result, and one test similar clinical effect to dapsone, but this was

48

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE 2 Showing the effects of various test sera on the rate of spread of intraderntatly injected dye Test Serum Source

1. Untreated scaile Dermatitis Herpeti-

Value of N

A Effect of Serum Cum pared with Effect ofHyaluronidase

12

Effect of Serum on the rate of Spread of

B Dye alone

C Dye plus ilyaluronidase

Less spreading

No effect

Accelerating effect

15

Less spreading

No effect

No effect

11

Less spreading

No effect

Accelerating effect

12

No difference

No effect

Accelerating effect

11

No difference

No effect

Accelerating effect*

21

Less spreading

No effect

No effect

21

Less spreading

Inhibitory No effect

formis

2. Prednisolone treated senile Dermatitis Herpetiformis

3. Untreated adult Dermatitis Herpetiformis (First case) 4. Untreated adult Dermatitis Herpetiformis (Second case)

5. Untreated adult Dermatitis Herpetiformis (Third case) 6. Dapsone treated Dermatitis Herpetiformis (First case) 7. Control case

effect

N = Number of tests carried out and compared. *

= This effect was just not sufficiently marked to be significant. TABLE 3 Showing the effects of various btister fluids and of hydrocortisone acetate on the rate of spread of intradermally injected dye

Test substance

A

Effect of Test sub-

Value

of N stance Compared with

Effect of ilyaluconidase

8. Blister fluid from adult Dermatitis Herpetiformis

Effect of Teat Substance on the Rate of Spread of

23

Less spreading

10 No difference

B Dye alone

No effect

C

Dye plus Hyaluronidase

Accelerating effect

blister fluid 10. Burn blister fluid Hydrocortisone Acetate

21

Less spreading

Accelerat- No effect ing effect No effect Accelerating effect

11. Alone

15

Less spreading

Inhibitory Inhibitory effect

9. Senile Dermatitis Herpetiformis

effect

4 No difference

12. Plus 9 above 13. Plus 10 above

12

Less spreading

No effect No effect

No effect No effect

N = Number of tests carried out and compared. not tested). When these therapeutic measures accelerates the spreading property of hyaluroniare withdrawn the disease relapses, which sug- dase during the first 40 minutes after the injection gests that the factor is not removed but its effect of this enzyme. This accelerating factor is in-

hibited by previous treatment with either

is only suppressed. 5UMMAET

A series of tests have been carried out which sltow that there is a factor present in the serum of

prednisolone or dapsone. Normal serum inhibits the effect of hyaluronidase.

Fluids from various blisters (from burns and dermatitis herpetiformis, which significantly dermatitis herpetiformis) were also tested but

HYALIJRONIDASE IN DERMATITIS HERPETIFORMIS

49

gave very inconsistent results from which no solutions for injection and for subsequently conclusions could be drawn. ACKNOWLEDGEMENTS

testing them for contamination. REFERENCES 1. SzonortAY, L.: Adatok a hyaluronidase-hyalu-

donsav osszefuggé-sckhcz pemphigusban da I am grateful to Dr. George Harvey for perdermatitis bcrpctiformis Duhringban. Bórmission to carry out these investigations in the gydgy Vencr Szcmle, 9: 46, 1955. J. C.: The influence of hormones front department of Dermatology of the Glasgow 2. OP5AIJL, the adrenal cortex on the dcrmal spread of Royal Infirmary. I am also indebted to Professor India ink with and without hyaluronidase. Yale J. Biol. Med., 21: 255, 1947—48. T. Symington for much helpful advice and critiL.: The role of ocdema in the clearance cism and for facilities in the Department of 3. JUELIN, of Evans Blue and hacmoglobin from inflam-

Pathology of the Glasgow Royal Infirmary to carry out the initial investigations on animals. I

should also like to thank Mr. W. MacCormack of the department of Bacteriology for preparing the

matory foci. Acta. Path. Microbiol. Scand., 38: 385,

1956. O'D.: The value of the eosinphil count in dermatitis herpetiformis. (to be pub-

4. ALEXANnEE,

lished)

J.

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94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.