(Plates LXVIII-LXXI)

Transcription

1 [GANN, 54, ; December, 1963] UDC : VIRUS-LIKE PARTICLES IN HUMAN CHLOROLEUKEMIA CELLS (Plates LXVIII-LXXI) Zensuke OTA, Shin-ya SUZUKI, and Satoru HIGASHI (Department of Internal Medicine, Okayama University Medical School*) Synopsis Unusual structures which suggested viral etiology of human leukemia were revealed by electron microscopy in bone marrow leukemic cells of a patient with chloroleukemia. These structures were as follows: 1) Intranuclear inclusion bodies essentially composed of relatively electron-dense fine granules and demarcated from the surrounding nucleoplasm by a clear zone. INTRODUCTION There is no doubt that viruses are causative agents of some of the malignant tumors in the animal kingdom. Experimental demonstration of such oncogenic viruses has been quite active in this field of research, particularly in mouse leukemias. It is, therefore, highly probable that viruses are also responsible for human leukemias. This paper is on an electron microscopic demonstration of intracytoplasmic viruslike particles and intranuclear inclusion bodies suggestive of a viral infection in a patient with chloroleukemia. Brief Note of the Clinical Course A 22-year-old male was admitted to our clinic on November 6, 1962, with chief complaints of headache, fever, and diplopia. He had had appendectomy 6 months prior to admission. In April of 1962, he gradually developed headache, fever, and then diplopia. He was treated in a hospital for possible tuberculous menigitis, but the symptoms and signs failed to improve. At that time, the cerebrospinal fluid contained blood. At the time of entry to our hospital, physical examination abducens paresis of the left eyeball, revealed positive Kernig's sign, rigidity of the neck, and motor and sensory paralysis of the extremities. Spinal puncture yielded a blooded cerebrospinal fluid. The cell count of the fluid was 245/mm3, 50% of which were neutrophilic promyelocytes (Table I, Photo 1). At that time, the peripheral blood and sternal aspiration showed no abnormal cells (Tables II and III). Our working diag- 481

2 Table I. Differential Count of Cells in the Cerebrospinal Fluid Table II. Blood Count of the Peripheral Blood Table III. Myeologram 482

3 nosis was menigitis due to leukemic infiltration. The reasons for this diagnosis were the appearance of immature leucocytes in the cerebrospinal fluid and the presence of asynchronism of nucleocytoplasmic development in the immature leucocytes. Therefore, administration of daily doses of 6mg of Myleran was started. Sternal aspiration performed on December 24, 1962, showed abundant proliferation of neutrophilic promyelocytes having the same morphologic characteristics as those in the cerebrospinal fluid (Photo 2). The patient died on January 20, The autopsy revealed the presence of a large green tumor in the sinus ethmoidalis posterior. Histologically the tumor was consistent with chloroma (Photo 3). The pathological diagnosis was chloroleukemia. MATERIAL AND METHODS The specimen used for electron microscopic observation was the aspirate of the second sternal puncture. Immediately after the aspirate was obtained, it was poured into the fixative3) which consisted of 1% osmic acid dissolved in Veronal-acetate buffer containing sucrose and adjusted to ph 7.2. The contact of the aspirate with were then dehydrated in a graded series of alcohols and embedded in a mixture of butyl and methyl methacrylates. Ultrathin sections were cut on a Leitz Ultramicrotome and mounted on a Formvar-covered grid. The sections were stained for 1 hour with uranyl acetate9) and examined in a Hitachi HU 10 electron microscope. EXPERIMENTAL RESULTS 1) Chloroleukemia Cells The electron microscopic findings of the majority of chloroleukemia cells are consistent with those of neutrophilic promyelocytes in the normal bone marrow (Photos 4, 5, 7, and 10). The nucleus is oval in shape, composed of diffusely scattered electrondense granules and the nuclear double membrane. The nucleoli are prominent. In the cytoplasm, the mitochondria and granules which vary in size, shape, and electron density are abundant. The compound vacuoles are occasionally seen. The roughsurfaced endoplasmic reticulum is well-developed. The lumen of the endoplasmic reticulum is enlarged and contains fine granules of low electron density. From the wall of the lumen, round protuberances are observed (Photo 5). Similar round structures are noticed within the lumens. These structures somewhat resemble mature virus particle, but their true nature is not clear. The smooth-surfaced endoplasmic reticulum is sparse. The Golgi body is large, having the well-developed Golgi canaliculi. The cytoplasm is rich in RNA granules, which occasionally form small aggregates. In some of the chloroleukemia nuclear region (Photo 10). cells, the fibrillar formation is seen at the peri- 483

4 2) Intranuclear Inclusion Bodies Intranuclear inclusion bodies are rather frequently observed in the chloroleukemia cells. There are three types of such inclusion bodies. a) A-Type inclusion bodies are composed of fine granules of moderate electron density which appeared to be arranged in a crystalline array. The granules are round is surrounded by a clear zone (Photo 6). Such inclusion bodies are also present in the nucleoli (Photo 7). b) B-Type inclusion bodies are essentially similar to the A-type inclusion body. However, the B-type inclusion body has a central core which is composed of fine 7 and 8). granules and an incomplete limiting membrane. From the surface of the inclusion body, filamentous structures project in an eddy fashion. The inclusion body is surrounded by a narrow clear zone (Photo 9). Similar inclusion bodies have been described in experimental malignant tumors of viral etiology and human malignant tumors.1,4,8) 3) Intracytoplasmic Virus-like Particles Four electron-dense ring-shaped particles are seen in the central region of a chloroleukemia cell (Photo 10). These particles are surrounded by RNA granules and an incomplete fibrillar formation, which is also seen at the perinuclear region. In Photo In the upper two particles, a double membrane is vague and a few small electrondense granules are seen on them. Around the particles, there are numerous moderately electron-dense fine particles, some of which appear filamentous. In the same micrograph, semicircular arrangements of RNA granules and a semicircular form of double membrane are seen. Within one of the semicircular forms of RNA granules, a portion of the double membrane is observed. These findings indicate that the particle and RNA granules are closely related, presumably the latter producing the former. In mammary cancer of the strain C3H mice5) and transplantable lymphoma of the Swiss mice,5) the intracytoplasmic A-type virus particles are round and composed of a double membrane. The particles described in the present paper appear to be very similar to these A-type virus particles in size and shape. Furthermore, structures resembling the particles observed are not known in human leucocytes. It is, therefore, probable that particles observed in the present preparation are intracytoplasmic virus particles. 484

5 DISCUSSION Although numerous investigations have been made, etiology of human leukemia remains unknown. Recent knowledges on experimental leukemia of viral etiology make us assume that human leukemia is also induced by a virus. Up to date, however, viral theory of human leukemia is only a hypothesis, since no experimental proof has been established. Dmochowski6,7) reported the presence of virus-like particles in lymph nodes of leukemic patients, but virus-cell relationship was not clear. Bessis1) described viruslike particles in human leukemia. However, these particles do not apper to satisfy the strict criteria of the morphology of virus particles. Some other descriptions2,10) on virus-like particles in human leukemia by means of electron microscopy seem to be unsatisfactory. The particles described in the present study are most likely virus particles present in a chloroleukemia cell, since they are composed of an electron-dense double membrane, closely resembling intracytoplasmic virus particles of experimental malignant tumors, and are not a known structure in human leucocytes. Relationship between the intranuclear inclusion bodies and the intracytoplasmic virus-like particles, both observed in the present study, is obscure. However, similar inclusion bodies have been observed in a number of malignant tumors of viral etiology.4,8) Therefore, the presence of intranuclear inclusion bodies in the chloroleukemia cell may also suggest a viral etiology of human leukemia. The authors express their sincere thanks to Prof. Kiyoshi Hiraki and Assist. Prof. Tadashi Ofuji for their helpful guidance and for reviewing this munuscript. The authors are also grateful to Mr. Noboru Kishimoto for his technical assistance. (Received August 24, 1963) REFERENCES 1) Bessis, M., Thiery, J. P., Nouvelle Rev. Franc. Hematol., 2, 577 (1962). 2) Braunsteiner, H., et al., Blood, 15, 476 (1960). 3) Caulfied, J. B., J. Biophys. Biochem. Cytol., 3, 827 (1957). 4) Claude, A., J. Ultrastruct. Res., 6, 1 (1962). 5) Dalton, A. J., Haguenau, F., "Tumors Induced by Viruses: Ultrastructural Studies," (1962). Academic Press Inc., New York. 6) Dmochowski, L., Grey, C. E., Texas Rept. Biol. Med., 15, 704 (1957). 7) Dmochowski, L., Science, 133, 551 (1961). 8) Homburger, F., "Progress in Tumor Research," (1963). S. Karger AG, Basel. 9) Watson, M. L., J. Biophys. Biochem. Cytol., 4, 475 (1958). 10) Zucker-Franklin, D., Blood, 21, 509 (1963). 485

6 EXPLANATION OF PLATES LXVIII-LXXI Photo 1. Cells in the cerebrospinal fluid. Photo 2. Sternal bone marrow cells. Photo 3. Histology of the tumor. Photo 4. A chloroleukemia cell having morphological characteristics of a normal neutrophilic promyelocyte. Photo 5. Chloroleukemia cells. Arrows indicate particles of unknown nature. Photo 6. Intranuclear A-type inclusion bodies (arrows) composed of moderately electron-dense fine granules in a crystalline array, surrounded by a clear zone. Photo 7. The upper arrow indicates a B-type inclusion body and the lower arrow, similar inclusion bodies. Photo 8. A partial enlargement of Photo 7. A core composed of electron-dense particles, in diameter, seen in the center of an inclcsion body in the nucleous. Photo 9. An intranuclear C-type inclusion body composed of fine granules and an incomplete limiting membrane. Fine filamentous structures projecting from the surface of the body. Photo 10. Arrows indicate virus-like particles, RNA granules, and the surrounding incomplete fibrillar formation. Photo 11. A partial enlargement of Photo 9. The lower two virus-like particles have a double membrane. The upper arrows indicate semicircular arrangements of RNA granules and a semicircular double membrane. Within one of the former (the upper left), a portion of the double membrane is seen. 486

7 GANN, Vol. 54 PLATE LXVIII

8 GANN, Vol. 54 PLATE LXIX

9 GANN, Vol. 54 PLATE LXX

10 GANN, Vol. 54 PLATE LXXI