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The Leishmania Test in Experimental Leishmaniasis (Oriental Sore) in Human Subjects1
THE LEISHMANIA TEST IN EXPERIMENTAL LEISHMANIASIS (ORIENTAL SORE) IN huMAN SUBJECTS' A. DOSTROVSKY, M.D.
The reaction of the skin to the intracutaneous injection of leishmania vaccine has been studied and established as a diagnostic test in animals by Wagener (1),
and in human subjects by Jessner and Amster (2a, b), Montenegro (3) and Dostrovsky (4). Results of further clinical and immunologic studies on the subject carried out in recent years were reported by Doubovskoy (5), Berberian (6), Dostrovsky (7) and Sagher (8a, b). The test constitutes a delayed reaction of the type of the tuberculin reaction. It is characterized by a high degree of specificity and sensitivity. Its sensitivity remains unchanged during the presence of the leishmania infection and even
after the lesions have healed. In our case, a positive test could be elicited as long as 30—40 years after a leishmania lesion had healed. The reactiOn in late forms of leishmaniasis (9) is particularly strong and sensitive. This evidence, in addition to the finding of Prausnitz-Kuestner antibodies in four of our cases, makes it certain that we are dealing with a reaction of microbial allergy (Dostrovsky and Sagher (10)). The following experiments constitute a further step in the clarification of the mechanism of this reaction. The object of these experiments is to ascertain the time of initial appearance of a positive reaction in an infected individual. Clinically, it is difficult to determine the precise moment when a Ieishinania infection
has taken place. We, therefore, undertook deliberate experimental infections of human subjects (volunteers among our clinical staff). TECHNIC
The procedure consisted of intracutaneous injections of from 2—16 millions of living flagellatae contained in 0.1—0.2 cc. of a leishmania tropica culture grown cn
N.N.N. agar, as an inoculation into the skin of the left upper arm, on its volar aspect. In cases Nos. 5 and 6 the inoculation was performed on the left thigh, for cosmetic reasons.
The leishmania test consisted in intracutaneous injection of leishmania vaccine into the skin of the volar aspect of the upper arms, viz: 0.1 cc. (1 million of kified fiagellatac per cc.) near the site of inoculation on the left, and another 0.1 cc. on the right upper arm. A control was done with intracutaneous injections of 0.1 cc. of phenolized normal saline. RESULTS
Case 1. The experimental subject was a 58 year old male, born in Europe, and had never had leishmania. One week prior to inoculation the leishmania test was negative. On 1
From the Dermatological Department (Head: Dr. A. Dostrovsky) of the Rothschild
Hadassah University Hospital, Jerusalem. Received for publication January 23, 1948. 435
436
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
January 23, 1945, he received an intracutaneous inoculation of 0.2 cc. (16 million living ftagellatae) of a leishmania culture into his left arm. SITE or INOCULATION
DATE
LEISHMANIA
LEST V.A.
RIGNT V.A.
1945
Jan. 24 Jan. 26 Jan. 28 Feb. 5 Feb. 8
Elevated area of erythema 4 cm. in diameter Same area now 1 cm. in diameter Erythematous infiltration 1 cm. in diameter Infiltration of 2 cm. in diameter Center softened, surrounded by red halo of 1
neg. pos.
—
—
+-i.
++
cm. wide. Microscopically, leishmania Feb. 11
parasites positive cc. of pus showed leishmania parasites microscopically and in cultures
pos.
+++ +++
Follow-up examination on December 15, 1947 (almost 3 years later) showed a leishmania scar at the site of inoculation and the leishmania test performed on that day gave a strongly positive reaction. Ca8e . The experimental subject was a 33 year old male who had never had a leishmania infection. One week prior to inoculation, his leishmania test was negative. On April 17, 1945, he was given an intracutaneous inoculation of a leishmania culture (0.1 cc. containing 2 million of fiagellatae) into the skin of his left upper arm. SITE OP INOCULATION
DATE
LEISEMANIA
LEST V.A.
RIGUT V.A.
1945
Apr. 18 Apr. 20
Apr. 22 Apr. 29 May 5
Elevated area of erythema of cm. in diameter Indurated, erythematous papule of a slightly bluish tinge Idem Nodule, bluish-red Ulcerated nodule, 3 cm. in diameter
neg. pos.
—
—
+
+
itching itching pos. pos.
+
+++
+
+++
Follow-up examination on December 15, 1947 (2 years later): an extremely positive response to the leishmania test could be elicited. Case S. The experimental subject was a 27 year old male who came from Europe and had never had a leishmania infection. One week prior to inoculation, his leishmania test was negative. On March 20, 1945, he received an intracutaneous inoculation of 4 millionflagellatae (0.1 cc.) at his left upper arm. SITE OF INOCULATION
DATE
LEISUMANIA
LEFT V.A.
EIGHT V.A.
1945
Mar. 20 Mar. 21 Mar. 22
Inoculation Erythematous swelling Induration in center of erythematous area cm. in diameter
neg. neg. pos.
++
Mar. 25 Apr. 9
Area of induration cm. in diameter
pos.
++
Area of induration 1 cm. in diameter, with ul-
ceration
++ +++
437
LEISHMANIA TEST IN HUMAN SUBJECTS
Follow-up examination on December 15, 1947 (2 years later) showed scarring at the site
of inoculation. The leishmania test performed on that day gave an intensely positive reaction.
Case 4. The experimental subject was a 37 year old male who came from Europe and had never had a leishmania infection. One week prior to inoculation his leishmania test was negative. On July 5, 1945, he received an intracutaneous inoculation of 0.2 cc. (3 million flagellatae)
into the skin of his left upper arm. DATE
SITE OF INOCVLATION
LEISEMANIA
LEFT V.A.
ELGET V.A.
1945
July 6 July 7 July 8 July 11
Elevated area of erythema 0.6 in diameter Area of infiltration of 1 cm. in diameter surrounded by an erythematous network Elevated area of 0.6 cm. in diameter; microscopically: leishmania parasites neg. More pronounced induration, otherwise unchanged. Microscopically and culturally, leishmania parasites positive.
neg.
pos. pos.
+ +
+ +
Case 5. The experimental subject was a 20 year old female who was born in Palestine and had never had leishmaniasis. One June 27, 1947, she was given an intracutaneous inoculation of 0.1 cc. of a leishmania culture containing 2 million flageilatae, into the skin of her left thigh. DATE
SITE OF INOCUI.ATION
LEISHMANIA TEST
1947
June 29
Brownish-red inoculation papule; elevated area 3 slightly pos.
July 1 July 7
cm. in diameter Idem p05. Indurated, brownish-red nodule of cm. in di- pos. ameter Idem strongly p05.
July 15
+ + ++
Case 6. The experimental subject was a 27 year old female from Europe and had never had leishmaniasis. One June 27, 1947, she was given an intracutaneous inoculation of 0.1 cc. of a leishmania culture containing 2 million fiageUatae into the skin of her left thigh. DATE
SITE OF INOCULATION
LEISEMANIA TEST
LEFT UPPER
1947
June 29
July 1 July 7 July 13
Reddish-brown papule cm. in diameter Nodule, cm. in diameter Indurated, bluish-red nodule Indurated, bluish-red nodule
neg. slightly pos. p05. strongly pos.
±
+
++
438
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY DISCUSSION
In the experiments described above, conducted on six experimental persons who could be carefully observed, the development of successful inoculations with graded doses of leishmania flagellatae as well as the first indication and further development of sensitizaton to the infection in the skin, could be followed and
observed at appropriate intervals. None of the persons on whom the experiments were performed had ever had a leishmania infection. The leishmania test performed intracutaneously with leishmania vaccine was negative before inoculation in four cases. In the other two it could only be performed two days after inoculation when it was also negative These cases, therefore, may also be regarded as having originally given negative responses. In all cases, the test performed either on the day of inoculation or two days later remained negative. Only the test performed 72 hours after inoculation was positive (after the lapse of another 24 and 48 hours). The skin sensitivity more or Iesspara1le1ed the
age of the inoculated lesion. (A test performed seven days after inoculation elicited a more pronounced skin reaction.) Positive reactions could be elicited even after the leishmania lesions had healed, as has been observed in "spontaneous" infections. However minute the inoculated lesion, the sensitivity produced was long-lasting and perhaps permanent. This could be demonstrated in follow-up tests in cases No. 1, 2, 3, 4 and 5. In all of these, a positive reaction could still be elicited after nearly three years. 'Whether in "spontaneous" leishmania infection the onset of skin sensitivity
also falls into so early a period, remains questionable. From the above, it appears that the reaction of sensitivity increases parallel to the age of the specific leishmania lesion. However, in our experiments this lesion was developed at a much earlier stage than in the case of "spontaneous" infection, since in the experiments—and this is a feature of particular interest—there was practically no incubation period in any of the test subjects. The reaction developing at the site of inoculation after 24 hours merged directly into the typical leishmania lesion (Dostrovsky (7)). The lesions thus produced were the result of introduction into the skin of doses
of the infective agent much more massive than in the case of "spontaneous" infection. The smallest dose used by us was 2 million fiagellatae as against 2000 flagellatae estimated by Adler (11) as being introduced into the skin in "spontaneous" infections. It is, therefore, probable that the slow development of the "spontaneous infection, as compared to the rapid development of the inoculated lesion, is reflected in the period of time needed for the development of skin sensitivity. Although both the lesion and the test reaction developed comparatively rapidly in our experiments, no difference in the intensity of the skin test could ascertained between areas close to and those remote from the site of inoculation. The earliest positive test (performed 72 hours after inoculation) showed no difference in its intensity (either 24 or 48 hours after its performance) between, the arm on which the inoculation had been made and the other arm, i.e., at a considerable distance from the site of inoculation. Whether, if examination could have been made more frequently, e.g., every hour, a difference would have been detected, is a question we are unable to answer.
LEISHMANIA TEST IN HUMAN SUBJECTS
439
SUMMARY
1. In six human volunteers who had never had a leishmania infection, leishmania lesions were produced by experimental inoculation.
2. The lesions developed rapidly, practically without incubation period, the inoculation reactions merging directly into typical leishmania lesions. 3. The extraordinary speed of the development of the lesions is best explained by the massive doses used for experimental infection.
4. The intracutaneous leishmania vaccine test was negative when performed on the day of inoculation, as well as one or two days later.
5. The first indication of skin sensitivity was observed in tests performed 72 hours after inoculation. 6. The older the lesion the more marked were the skin reaction to leishmania vaccine.
7. A positive test could be elicited during all the time the lesion was present and during a follow-up period of three years even when the lesions of inoculation had completely healed.
8. Neither the intensity nor the time of appearance of the skin test reaction could be observed to differ between areas close to and those at a greater distance from the site of experimental inoculation. REFERENCES 1. WAGENER, E. H.: A skin reaction to extracts of Leishmania Tropica and Leishmania Infantum. Univ. California Pubi. Zool., 20: 477—488, 1923. 2. JESSNER, M. AND AMSTER, S.: (a) Leishmania vaccine—Leishmaniinreaction. Deutsche med. Wchnschr., 61: 784—785, (May 8) 1925. (b) JE5SNER, M.: Untersuchungen ueber die Wirkung von Leishmaniavaccine bei experimenteller Hautleishmaniose. Arch. 1. Dermat. u. Syph., 153: 237—247, 1927.
3. MONvENEGRO, J.: Cutaneous reaction in leishmaniasis. Arch. Dermat. & Syph., 13: 187—194 (Feb.) 1926. 4. DosmovsY, A.: The diagnostic value of leishmania vaccine. Ann. Trop. Med., 29:
123—128 (July) 1935.
5. Douovsxor, P. A.: An intradermal allergic reaction in cutaneous leishmaniasis. In "Problems of Cutaneous Leishmaniasis", Ashkhabad, 1941. Quoted from bARE, C. A.: Cutaneous leishmaniasis: critical review of recent Russian work. Trop. Dis. Bull., 41: 331—345 (May) 1944.
6. BERBERIAN, D. A.: Cutaneous leishmaniasis (oriental sore). II. Incubation period. Arch. Dermat. & Syph., 50: 231—232 (Oct.) 1944.
7. Dosrxovany, A.: The incubation period in experimental cutaneous leishmaniasis. Acta med. orient., 4: 303—305 (Sept.) 1945.
8. SAGHER, F.: (a) Activation of an originally negative intracutaneous Leishinania vaccine reaction. Acta med. orient., 5: 82—85 (March) 1946. (b) The response of scars of cutaneous leishmaniasis to the injection of Leishmania Tropica vaccine. Brit. J. Dermat. 59: 205—213 (June) 1947.
9. SAGHER, F.: Leishmania vaccine test in leishmaniasis of the skin (oriental sore). Quantitative experiments. Arch. Dermat. & Syph., 55: 658—663 (May) 1947. 10. DOSTROVSKY, A. AND SAGRER, F.: The intracutaneous test in cutaneous leishmaniasis. Ann. Trop. Med., 40: 265—269 (Dec.) 1946.
11. Arn.Ei, S.: Personal communication.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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.
The reaction of the skin to the intracutaneous injection of leishmania vaccine has been studied and established as a diagnostic test in animals by Wagener (1),
and in human subjects by Jessner and Amster (2a, b), Montenegro (3) and Dostrovsky (4). Results of further clinical and immunologic studies on the subject carried out in recent years were reported by Doubovskoy (5), Berberian (6), Dostrovsky (7) and Sagher (8a, b). The test constitutes a delayed reaction of the type of the tuberculin reaction. It is characterized by a high degree of specificity and sensitivity. Its sensitivity remains unchanged during the presence of the leishmania infection and even
after the lesions have healed. In our case, a positive test could be elicited as long as 30—40 years after a leishmania lesion had healed. The reactiOn in late forms of leishmaniasis (9) is particularly strong and sensitive. This evidence, in addition to the finding of Prausnitz-Kuestner antibodies in four of our cases, makes it certain that we are dealing with a reaction of microbial allergy (Dostrovsky and Sagher (10)). The following experiments constitute a further step in the clarification of the mechanism of this reaction. The object of these experiments is to ascertain the time of initial appearance of a positive reaction in an infected individual. Clinically, it is difficult to determine the precise moment when a Ieishinania infection
has taken place. We, therefore, undertook deliberate experimental infections of human subjects (volunteers among our clinical staff). TECHNIC
The procedure consisted of intracutaneous injections of from 2—16 millions of living flagellatae contained in 0.1—0.2 cc. of a leishmania tropica culture grown cn
N.N.N. agar, as an inoculation into the skin of the left upper arm, on its volar aspect. In cases Nos. 5 and 6 the inoculation was performed on the left thigh, for cosmetic reasons.
The leishmania test consisted in intracutaneous injection of leishmania vaccine into the skin of the volar aspect of the upper arms, viz: 0.1 cc. (1 million of kified fiagellatac per cc.) near the site of inoculation on the left, and another 0.1 cc. on the right upper arm. A control was done with intracutaneous injections of 0.1 cc. of phenolized normal saline. RESULTS
Case 1. The experimental subject was a 58 year old male, born in Europe, and had never had leishmania. One week prior to inoculation the leishmania test was negative. On 1
From the Dermatological Department (Head: Dr. A. Dostrovsky) of the Rothschild
Hadassah University Hospital, Jerusalem. Received for publication January 23, 1948. 435
436
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
January 23, 1945, he received an intracutaneous inoculation of 0.2 cc. (16 million living ftagellatae) of a leishmania culture into his left arm. SITE or INOCULATION
DATE
LEISHMANIA
LEST V.A.
RIGNT V.A.
1945
Jan. 24 Jan. 26 Jan. 28 Feb. 5 Feb. 8
Elevated area of erythema 4 cm. in diameter Same area now 1 cm. in diameter Erythematous infiltration 1 cm. in diameter Infiltration of 2 cm. in diameter Center softened, surrounded by red halo of 1
neg. pos.
—
—
+-i.
++
cm. wide. Microscopically, leishmania Feb. 11
parasites positive cc. of pus showed leishmania parasites microscopically and in cultures
pos.
+++ +++
Follow-up examination on December 15, 1947 (almost 3 years later) showed a leishmania scar at the site of inoculation and the leishmania test performed on that day gave a strongly positive reaction. Ca8e . The experimental subject was a 33 year old male who had never had a leishmania infection. One week prior to inoculation, his leishmania test was negative. On April 17, 1945, he was given an intracutaneous inoculation of a leishmania culture (0.1 cc. containing 2 million of fiagellatae) into the skin of his left upper arm. SITE OP INOCULATION
DATE
LEISEMANIA
LEST V.A.
RIGUT V.A.
1945
Apr. 18 Apr. 20
Apr. 22 Apr. 29 May 5
Elevated area of erythema of cm. in diameter Indurated, erythematous papule of a slightly bluish tinge Idem Nodule, bluish-red Ulcerated nodule, 3 cm. in diameter
neg. pos.
—
—
+
+
itching itching pos. pos.
+
+++
+
+++
Follow-up examination on December 15, 1947 (2 years later): an extremely positive response to the leishmania test could be elicited. Case S. The experimental subject was a 27 year old male who came from Europe and had never had a leishmania infection. One week prior to inoculation, his leishmania test was negative. On March 20, 1945, he received an intracutaneous inoculation of 4 millionflagellatae (0.1 cc.) at his left upper arm. SITE OF INOCULATION
DATE
LEISUMANIA
LEFT V.A.
EIGHT V.A.
1945
Mar. 20 Mar. 21 Mar. 22
Inoculation Erythematous swelling Induration in center of erythematous area cm. in diameter
neg. neg. pos.
++
Mar. 25 Apr. 9
Area of induration cm. in diameter
pos.
++
Area of induration 1 cm. in diameter, with ul-
ceration
++ +++
437
LEISHMANIA TEST IN HUMAN SUBJECTS
Follow-up examination on December 15, 1947 (2 years later) showed scarring at the site
of inoculation. The leishmania test performed on that day gave an intensely positive reaction.
Case 4. The experimental subject was a 37 year old male who came from Europe and had never had a leishmania infection. One week prior to inoculation his leishmania test was negative. On July 5, 1945, he received an intracutaneous inoculation of 0.2 cc. (3 million flagellatae)
into the skin of his left upper arm. DATE
SITE OF INOCVLATION
LEISEMANIA
LEFT V.A.
ELGET V.A.
1945
July 6 July 7 July 8 July 11
Elevated area of erythema 0.6 in diameter Area of infiltration of 1 cm. in diameter surrounded by an erythematous network Elevated area of 0.6 cm. in diameter; microscopically: leishmania parasites neg. More pronounced induration, otherwise unchanged. Microscopically and culturally, leishmania parasites positive.
neg.
pos. pos.
+ +
+ +
Case 5. The experimental subject was a 20 year old female who was born in Palestine and had never had leishmaniasis. One June 27, 1947, she was given an intracutaneous inoculation of 0.1 cc. of a leishmania culture containing 2 million flageilatae, into the skin of her left thigh. DATE
SITE OF INOCUI.ATION
LEISHMANIA TEST
1947
June 29
Brownish-red inoculation papule; elevated area 3 slightly pos.
July 1 July 7
cm. in diameter Idem p05. Indurated, brownish-red nodule of cm. in di- pos. ameter Idem strongly p05.
July 15
+ + ++
Case 6. The experimental subject was a 27 year old female from Europe and had never had leishmaniasis. One June 27, 1947, she was given an intracutaneous inoculation of 0.1 cc. of a leishmania culture containing 2 million fiageUatae into the skin of her left thigh. DATE
SITE OF INOCULATION
LEISEMANIA TEST
LEFT UPPER
1947
June 29
July 1 July 7 July 13
Reddish-brown papule cm. in diameter Nodule, cm. in diameter Indurated, bluish-red nodule Indurated, bluish-red nodule
neg. slightly pos. p05. strongly pos.
±
+
++
438
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY DISCUSSION
In the experiments described above, conducted on six experimental persons who could be carefully observed, the development of successful inoculations with graded doses of leishmania flagellatae as well as the first indication and further development of sensitizaton to the infection in the skin, could be followed and
observed at appropriate intervals. None of the persons on whom the experiments were performed had ever had a leishmania infection. The leishmania test performed intracutaneously with leishmania vaccine was negative before inoculation in four cases. In the other two it could only be performed two days after inoculation when it was also negative These cases, therefore, may also be regarded as having originally given negative responses. In all cases, the test performed either on the day of inoculation or two days later remained negative. Only the test performed 72 hours after inoculation was positive (after the lapse of another 24 and 48 hours). The skin sensitivity more or Iesspara1le1ed the
age of the inoculated lesion. (A test performed seven days after inoculation elicited a more pronounced skin reaction.) Positive reactions could be elicited even after the leishmania lesions had healed, as has been observed in "spontaneous" infections. However minute the inoculated lesion, the sensitivity produced was long-lasting and perhaps permanent. This could be demonstrated in follow-up tests in cases No. 1, 2, 3, 4 and 5. In all of these, a positive reaction could still be elicited after nearly three years. 'Whether in "spontaneous" leishmania infection the onset of skin sensitivity
also falls into so early a period, remains questionable. From the above, it appears that the reaction of sensitivity increases parallel to the age of the specific leishmania lesion. However, in our experiments this lesion was developed at a much earlier stage than in the case of "spontaneous" infection, since in the experiments—and this is a feature of particular interest—there was practically no incubation period in any of the test subjects. The reaction developing at the site of inoculation after 24 hours merged directly into the typical leishmania lesion (Dostrovsky (7)). The lesions thus produced were the result of introduction into the skin of doses
of the infective agent much more massive than in the case of "spontaneous" infection. The smallest dose used by us was 2 million fiagellatae as against 2000 flagellatae estimated by Adler (11) as being introduced into the skin in "spontaneous" infections. It is, therefore, probable that the slow development of the "spontaneous infection, as compared to the rapid development of the inoculated lesion, is reflected in the period of time needed for the development of skin sensitivity. Although both the lesion and the test reaction developed comparatively rapidly in our experiments, no difference in the intensity of the skin test could ascertained between areas close to and those remote from the site of inoculation. The earliest positive test (performed 72 hours after inoculation) showed no difference in its intensity (either 24 or 48 hours after its performance) between, the arm on which the inoculation had been made and the other arm, i.e., at a considerable distance from the site of inoculation. Whether, if examination could have been made more frequently, e.g., every hour, a difference would have been detected, is a question we are unable to answer.
LEISHMANIA TEST IN HUMAN SUBJECTS
439
SUMMARY
1. In six human volunteers who had never had a leishmania infection, leishmania lesions were produced by experimental inoculation.
2. The lesions developed rapidly, practically without incubation period, the inoculation reactions merging directly into typical leishmania lesions. 3. The extraordinary speed of the development of the lesions is best explained by the massive doses used for experimental infection.
4. The intracutaneous leishmania vaccine test was negative when performed on the day of inoculation, as well as one or two days later.
5. The first indication of skin sensitivity was observed in tests performed 72 hours after inoculation. 6. The older the lesion the more marked were the skin reaction to leishmania vaccine.
7. A positive test could be elicited during all the time the lesion was present and during a follow-up period of three years even when the lesions of inoculation had completely healed.
8. Neither the intensity nor the time of appearance of the skin test reaction could be observed to differ between areas close to and those at a greater distance from the site of experimental inoculation. REFERENCES 1. WAGENER, E. H.: A skin reaction to extracts of Leishmania Tropica and Leishmania Infantum. Univ. California Pubi. Zool., 20: 477—488, 1923. 2. JESSNER, M. AND AMSTER, S.: (a) Leishmania vaccine—Leishmaniinreaction. Deutsche med. Wchnschr., 61: 784—785, (May 8) 1925. (b) JE5SNER, M.: Untersuchungen ueber die Wirkung von Leishmaniavaccine bei experimenteller Hautleishmaniose. Arch. 1. Dermat. u. Syph., 153: 237—247, 1927.
3. MONvENEGRO, J.: Cutaneous reaction in leishmaniasis. Arch. Dermat. & Syph., 13: 187—194 (Feb.) 1926. 4. DosmovsY, A.: The diagnostic value of leishmania vaccine. Ann. Trop. Med., 29:
123—128 (July) 1935.
5. Douovsxor, P. A.: An intradermal allergic reaction in cutaneous leishmaniasis. In "Problems of Cutaneous Leishmaniasis", Ashkhabad, 1941. Quoted from bARE, C. A.: Cutaneous leishmaniasis: critical review of recent Russian work. Trop. Dis. Bull., 41: 331—345 (May) 1944.
6. BERBERIAN, D. A.: Cutaneous leishmaniasis (oriental sore). II. Incubation period. Arch. Dermat. & Syph., 50: 231—232 (Oct.) 1944.
7. Dosrxovany, A.: The incubation period in experimental cutaneous leishmaniasis. Acta med. orient., 4: 303—305 (Sept.) 1945.
8. SAGHER, F.: (a) Activation of an originally negative intracutaneous Leishinania vaccine reaction. Acta med. orient., 5: 82—85 (March) 1946. (b) The response of scars of cutaneous leishmaniasis to the injection of Leishmania Tropica vaccine. Brit. J. Dermat. 59: 205—213 (June) 1947.
9. SAGHER, F.: Leishmania vaccine test in leishmaniasis of the skin (oriental sore). Quantitative experiments. Arch. Dermat. & Syph., 55: 658—663 (May) 1947. 10. DOSTROVSKY, A. AND SAGRER, F.: The intracutaneous test in cutaneous leishmaniasis. Ann. Trop. Med., 40: 265—269 (Dec.) 1946.
11. Arn.Ei, S.: Personal communication.
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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.