Acta Radiologica: Diagnosis

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Irradiation Effects Upon the Fetal Central Nervous System of Macacus Rhesus Monkeys: Effects on lysosomes L Roizin & M. A. Kaufman To cite this article: L Roizin & M. A. Kaufman (1966) Irradiation Effects Upon the Fetal Central Nervous System of Macacus Rhesus Monkeys: Effects on lysosomes, Acta Radiologica: Diagnosis, 5:1, 161-176 To link to this article: http://dx.doi.org/10.3109/02841856609139554

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Date: 17 January 2017, At: 00:20

FROM T H E DEPARTMENT OF NEUROPATHOLOGY, N.Y.S. THE

DEPARTMENT

OF

RADIOLOGY,

RADIOLOGICAL

PSYCHIATRIC INSTITUTE,

RESEARCH

LABORATORIES,

AND T H E DEPARTMENT OF PATHOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, N.Y., U.S.A.

IRRADIATION EFFECTS UPON THE FETAL CENTRAL NERVOUS SYSTEM OF MACACUS RHESUS MONKEYS Effects on lysosomes

L. ROIZIN,R. RUGHand M. A. KAUFMAN Our previous experimental investigations dealt with the effects of roentgen irradiation upon the central nervous system of rat embryos irradiated in utero at 8.5, 9.5, 15.5 and 16.5 gestation days. Correlated gross, histopathologic and electron microscopic studies revealed a variety of maldevelopments or dysplasias of the central nervous system. Some of these, at times, were preceded by or associated with various degrees of cellular, ultrastructural and necrobiotic alterations (29, 30). The present study is concerned with the fetal central nervous system of the Macacus rhesus monkey following intra-uterine roentgen irradiation. These investigations aim in particular at exploring possible pathogenetic mechanisms related to the necrobiotic processes. Since certain general findings were similar to some of our previously reported studies and because of space limitation, in the present paper we will consider principally ( 1) the possible role that lysosomes may play during some phases of the necrobiotic processes, (2) their significance 11 4 5 3 2 7 2 . Acta Radiologica Ther. Phys. Biol.

5 (1966)

161

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DAYS

Fig. 1. Composite graph showing related weight and crown-rump length of Macaca mulatta monkey according to VAN WACENEN& ASLING. (Courtesy Dr. G. Van Wagenen.)

in initiating a pathogenetic chain of tissue reactions of an autosensitization character which ultimately leads to (3) hyperergic tissue reactions and necrotizing lesions through an iso-allergic mechanism similar to that in experimental allergic encephalomyelitis. The monkeys (Macacus Mullata, popularly called Rhesus Macaque) were obtained by air express directly from India, impregnated in nature, While the exact time of impregnation could never be determined, upon arrival at the laboratory each monkey was rectally examined to determine whether it was pregnant, and a rough estimate made as to the approximate size of the fetus. The gestation period of this monkey is 150 to 160 days and the youngest fetus used in this series was approximately 70 days of age at the time of irradiation. The estimates of age were based upon recent statistical studies by VAN WAGENEN & ASLING(34) and were related to fetal weight and crown-rump length. A composite graph of these data is given in Fig. 1. I n most cases, the

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Table 1 Number

of monkeys, radiation

dosage, post-irradiation interval and age at sacrifice

Monkey No.

Dosage

5 6 11 13 14 16 20 22 23 41 46 52 53 44 49

260 260 265 200 130 Control 200 Control 200 195 195 500 600 Control 400

(L-5152) (L-5153) (L-5196) (L-5197) (L-5226) (L-5192) (L-5234) (L-5235) (L-5271) (L-5468) (L-5469) (L-5492) (L-5493) (L-5505) (L-5501)

R

Post-irradiation interval

Age a t sacrifice

20 20 45 45 22

110-120 g.d. 130 g.d. Full term Full term 1 day old Full term 13 days old 13 days old 12 days old 90 g.d. 88 g.d. Full term Full term Full term 90 g.d.

hrs hrs hrs hrs days

-

19 days -

5 weeks 3 weeks 3 weeks 24 hrs 24 hrs -

48 hrs

monkey fetuses were allowed to come to term and were sacrificed at various times thereafter (Table I). In these cases it was a simple matter to calculate back to the time of irradiation and thus determine the approximate age at exposure. I n those instances where sacrifice was made prior to delivery, the calculations were based entirely upon the crown-rump length and body weight data. A total of 12 irradiated and three control monkey fetuses were involved in this study, five of those irradiated being sacrificed before birth. Monkey fetal material is difficult to acquire and yet extremely valuable in investigating primate radiation-reactions. Since in every instance of this study the fetuses or newborn were apparently in otherwise normal health, the data, even from these few examples, is considered significant. The radiation facilities used were as follows: a single roentgen beam was passed through a 3 inch diameter lead cone at a distance of 49.5 cm to the estimated center of the gravid uterus with the (nembutal) anesthetized monkey lying on its side; with a skin air dose of 100 R/min tile depth dose estimate of 70 yo of the air dose at that position of the fetus (70 Rimin), with half of the prescribed dose delivered and then the monkey turned onto its other side

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and the fetus irradiated with the second half of the prescribed dose. I n this way there was even distribution of the ionizations through the tissues of the fetus. The roentgen apparatus was run a t 184 kV, 30 mA, with 0.28 mm Cu and 0.50 mm A1 filtration giving an HVL of 0.6 mm Cu. The dose rate was 50 R/min to the fetus. Following exposure the pregnant monkey was returned to its cage and given abundant food and water, supplemented by milk. I n the instances when the fetus was removed from the uterus prior to delivery, this procedure was carried out by laparotomy under nembutal anesthesia with an excess of 2 ml subcutaneous injection. Biopsies for electron microscope investigations were taken from various regions of the cerebrum, cerebellum and spinal cord while the animals were still alive. The biopsy material was immediately fixed in Palade’s buffered osmic tetroxide and Dalton’s fixatives, gradually dehydrated, embedded in methacrylate or Epon media, sectioned with LKB Ultratome, treated with KMmO, or uranyl acetate and examined with RCA, EMU-2B or 3G electron microscope. The remaining central nervous system material was partially fixed in 10 % isotonic neutral formalin and 80 yo alcohol for histologic studies. These included Nissl, and hematoxylin and eosin stains for cytologic studies, Sudan I11 for fat stainable material, Spielmeyer for myelin and the periodic acid-Schiff reaction (PAS) for mucopolysaccharides, Electron microscope studies of lysosomes

Thefine structural and cytochemical properties. The lysosomes are a heterogenous group of intracytoplasmic organelles first described by DE DUVEet coll. (5, 6 ) . The name ‘Iysosomes’ was proposed by these authors for a group of ‘intracytoplasmic particles’ which originally were associated with five distinct acid hydrolases. Subsequently the cytological identity of hepatic lysosomes was confirmed by electron microscope (26), the application of the Gomori acid phosphatase procedure to frozen sections (15, 22,23) and the examination of acid phosphatase preparations in the electron microscope (3, 10, 16). For more detailed references, 20, 24, 25 should be consulted. I n brief, the lysosomes are a heterogenous group of intracytoplasmic organelles surrounded by a single membrane-like barrier which walls off the internal enzymes and structure. Metabolic and functional correlations. The metabolic activities of lysosomes in vivo and in vitro are summarized in Table 2. I t is significant, however, that although originally (5, 6) it was suggested that all the lysosomal enzymes have an acid p H optimum, it must be kept in mind that most cells also contain hydrolases with a neutral or alkaline optimum (4). Some authors have used acid phosphatase activity as a ‘lysosomal tracer’ or

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Table 2 Structural, metabolic functional and pathologic correlations of lysosomes in in uivo and in oiho conditions Structure metabolic activities

Functional properties

Pathological reactions

In vivo

Acid phosphatase /3-glucuronidase LC. R-Nase, ac. D-Nase ac-protease(s), cathepsin(s) /3-N-acetylglucoseaminadase @-galactosidase Aryl sulfatase Lysozyme (WBC) Protein-bound glycolipids

Homeokinetic Endogenous digestion Phagocytosis Pinocytosis Arthrocytosis

Metamorphosis Abiotrophy Autolysis Necrobiosis Allergic processes

In nitro

Same as above

Various degrees as above Autolysis and/or necrosis (tissue culture)**

*

* Experimental

injuries: autolysis, ischemia, hypervitaminosis A, hypovitaminosis E, phagocytosis (WBC), endotoxine, traumatic shock, U. V. and roentgen irradiations. ** Experimental injuries: freezing and thawing, Waring blender, non-ionic detergents vitamin A., U. V. irradiation, hypo-osmolar conditions.

‘marker’ (5, 27, etc.). Although their origin is still disputed, according to some authors (27, 33) it is assumed that: a) the acid phosphatase is elaborated in the endoplasmatic reticulum and concentrated in the Golgi vesicles which coalesce to form digestive vacuoles which in turn incorporate the pinocytotic vacuoles or foci of degradation (endogenous digestion, active form of cytoplasmic degradation or autophagia), and b) the acid phosphatase appears by local concentration of preexisting hydrolases in an area of digestion or degradation (passive form of cytoplasmic degradation, 33). I t seems, on the basis of electron microscope observations that the lysosomes are not just simple bags filled with various types of enzymes, but that they might also contain non-enzymatic constituents. Among the latter, thus far have been detected phospholipids and PAS positive material (liver, kidney, 24) protein-bound glycolipids (nervous tissue, 19), iron hemosiderin-like material, and lipofuscin (macrophages, liver parenchyma and perhaps other cells, 19), and fluorescent vacuoles (24). I t is important to recall that lysosomes display qualitative as well as quantitative heterogeneity in the sense that their morphologic appearance, enzymatic properties and concentrations may vary not only in different species of animals, but may also vary from one organ to another and even in the same tissue. Al-

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K. RUGH A N D M. A . KAUFMAN

Fig. 2. Electron microgram from RCA-EMU-PB, monkey 16. Cerebral cortex of control monkey illustrating the average appearance of lysosomes. Magnification 78 400 x .

though during recent years a remarkable number of new observations and new biochemical, metabolic and functional hypotheses have generated from the in vitro and in vivo studies of lysoso111cs, it still remains for the future to clarify the origin of the lysosomes, as well as the growing number of their enzymatic contents and the respective biochemical and functional correlates.

Pathologic aspects. I n relation to our topic of discussion, on the relationship of lysosomes and X-ray necrobiosis, it is of particular significance to mention that lysosomal permeability, perforation, rupture, etc., as a possible cause of cell damage and cell death was the first pathological implication of the Iysosome concept (5, 6). This may result from a variety of causes as indicated in the last column of Table 2. I n other words, structurally damaged or altered lysosomal membrane barriers would release enzyme systems which would induce uncontrollable lysis of the surrounding cellular elements. Consequently, the involved tissue constituents will undergo disorganization and degradation with separation and liberation of products or material of disintegration. The latter will appear as residual material or electron opaque material in electron microscope terminology. Their chemical composition is of a variable character in accord with the enzymatic effects. I t appears also possible that the intensity or severity of these alterations will be correlated with the quantitative and qualitative content of the lysosome. For instance, HIRCH& COHN(14) have shown that preparations of WBC lysosomal granules in vitro exhibited maximal enzymatic activity in their various substrates only after disruption by physical et coll. (35) injected streptolysin and chemical means. I n addition, WEISSMAN S (the non-antigenic toxin) in the knee joints of rabbits in order to determine

IRRADIATION EFFECTS UPON FETAL NERVOUS SYSTEM OF MONKEYS

a

167

b

Fig. 3. Electron micrograms from RCA-EMU-ILB, monkey 14 (a) (magnification 78 400 X ), and monkey 13 (b) (magnification 396 900 x ). Various degrees of alterations of lysosomes in thc cortex of post-irradiated monkeys.

whether damage to lysosomes of tissue in the living animal could lead to acute or chronic tissue injury. I n each of the 108 injected joints an inflammatory reaction was induced and the morphologic changes of synovial lysosomes were demonstrated by the acid phosphatase stains (GOMORI). Furthermore, in threefourths of the animals developing a chronic arthritis, circulating complimentfixing antibodies directed against the membranes of lysosomes, but not against mitochondria, lysosomes or cell supernatant fractions of homogenate from heterologous or homologous liver were demonstrated. These observations indicate that a chronic form of inflammatory injury had elicited auto-antibodies against antigenic determinants unmasked as a result of lysosomal injury.

Electron microscopic findings in the irradiated cerebral tissue. Up to date, studies have revealed that of the 12 prenatally irradiated monkeys, 5 have shown various degrees of lysosomal alterations (monkeys: 11, 13, 14, 20, 23), some of which also showed inflammatory (monkeys 20, 52, 53) and necrobiotic

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L. ROIZIN, R. KUGH AND M. A. KAUFMAN

a

b

Fig. 4. Electron micrograms from RCA-EMU-ZB, inonkey 13. Cerebral cortex of the post-irradiated animal showing variable granular and particulate material of different electron density within the matrix of lysosomes (a) (magnification 78 400 x ), and cytolysosomes (b) (magnification 235 200 x ).

reactions (monkeys: 13, 14). Fig. 2 illustrates the average fine features of lysosomes in the cerebral cortex as revealed by the electron microscope, i.e. roundish or ovoidal organelles of variable size (average 0.4 p) consisting of a more or less regularly dense osmiophilic matrix, surrounded by a single membrane through which biochemical and functional environmental exchanges are taking place with the surrounding cytoplasm or structural constituents. Fig. 3 shows marked variations in the fine structural matrix in its osmiophilic character; in Fig. 3 a the electron opaque material appears markedly dense and within it some denser particulate products as well as three vacuoles are noted, whereas in Fig. 3 b the matrix is composed mostly of irregular granular and dust-like products. Some variations in the osmiophilic character, vacuoles (two) and alterations in the finest texture of the matrix are also noted. In addition irregularities and lack of differentiation in Fig. 3 a as well as disruption of the limiting membrane in Fig. 3 b are evident. In some instances (Fig. 4 a) the lysosomal matrix also appeared disorganized and disrupted by particulate, dense osmiophilic products of various shapes, sizes or configurations. Furthermore, at times (Fig. 4 b) the lysosomes appeared

IRRADIATION EFFECTS UPON FETAL NERVOUS SYSTEM OF MONKEYS

Fig. 5. Electron micrograms from RCA-EMU-2B, magnifications 78 400 x . Upper view: Monkey 20. A mononuclear cell. Lower view: Monkey 14. A polymorphonuclear granulocyte attached to the vascular wall.

169

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abnormally enlarged with variable disrupted or disorganized matrix and limiting membranes, and contained vacuoles of vesicles as well as different residual-like bodies, and assumed the appearance of cytolysomes (24). Of interest is the fact that in some of the examined areas, blood vessels appeared to be involved or surrounded by various degrees of inflammatory exudate as is partially illustrated in Fig. 5. I n these circumstances, at times, were also observed the presence of some osmiophilic bodies or some composite structures containing variably dense osmiophilic material intermixed with vacuoles or pinocytotic vesicles. On still other occasions (Fig. 6, top left) groups of lysosomes were seen undergoing various degrees of alterations and disruptions giving the impression that some of the osmiophilic matrix was spilling into the surrounding tissues. I n some instances, the individual lysosomes were difficult to identify as they involved tissues displaying conglomerations of variable osmiophilic products intermixed or trapped with membranous structures and at times multilaminated formations of myelin-like figures (Fig. 6, top right). I n addition, in some areas more severe tissue alterations were encountered which gave the impression of various phases of pre- and/or necrotizing lesions (Fig. 6, lower view). I t is also of particular significance to mention that histologic stains or lipid material, myelin sheath, and Gomori’s acid phosphatase methods showed in some areas various degrees of intra- and extra-cellular tissue alterations. Finally, PAS stains showed strong positive granular reactions. The latter findings will be reported separately.

Discussion O n the basis of the afore-mentioned observations it is suggested that (1) there is evidence, in certain areas of the prenatally roentgen irradiated monkey central nervous system, of concomitant occurrence of fine structural alterations of lysosomes (Figs 3 and 4), inflammatory reactions (Fig. 5), and pre- and/or necrobiotic processes (Fig. 6) and (2) the latter processes may initiate a chain of autoimmune mechanisms which may induce autosensitization and isoallergic reactions. Although this hypothetical relationship is being studied with additional experimental material, histologic and histochemical methods, and needs further confirmation, nevertheless this possibility is also suggested by the following similar investigations reported by several authors as well as by us.

Lysosomes and necrobiotic processes. The possibility of lysosomal damage (perforation or rupture) as a cause of cellular injury and necrosis was considered among the first pathological consequences of the lysosomal concept (5, 6).

IRRADIATION EFFECTS UPON FETAL NERVOUS SYSTEM OF MONKEYS

Fig. 6. Electron micrograms from RCA-EMU-ZB, monkey 23. Various degrees of lysosomal alterations in areas undergoing structural disorganization and degradation and some necrosis. Upper left: magnification 205 800 x ,upper right: magnification 78 400 x , lower view: magnification 205 800 x .

I71

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Additional data in vivo and in vitro studies are summarized under column ‘pathological reactions’ in Table 2.

Lysosomes and effects of irradiations on the central neruous system. In our previous studies concerning the effects of irradiation of rat embryo brains, we have shown various degrees of quantitative and qualitative changes of lysosomes particularly in association with pre- and necrobiotic processes (29, 30). Lysosomal damage following irradiations was also observed by several investigators in the spleen (31), lymphatic cells (21), liver (32), and neurons (following whole body irradiation, 18). Lysosomes in immunologic processes. To date, studies reveal that lysosomes which could be identified in widely diversified cells react to alter cellular physiology either by changes in the membrane permeability to substrates, or release of hydrolases into intracellular vacuoles and to intracellular environment (7). However, the stability of fragility of the lysosome membrane showed marked variations in various types of cells in tissues of animals (2). Since the stability of lysosomal membranes was not constant and could change within physiological conditions, the cytologic effect of antibody and complement on ascites tumor cells was selected in view of the fact that, 1) it is performed on isolated cells under fully controlled conditions, and 2) a process of necrosis could be followed from the time the cells are exposed to damaging agents until the final et coll. (9) necrotic state (2). For instance, in such circumstances DUMONDE observed increase in the fragility of lysosomes and swollen cells before there was evidence of cytolysis. I t was also shown by fluorescent technique that the antibody was bound only to the antigenic side on the surface of the cell membrane, and that in the absence of the complement, the antibody was not able to penetrate to the cell interior (8). I n order not to deviate from the main purpose of our discussion, the interested reader is referred to bibliographical references 1 and 2. Here we would mention only briefly that it is possible to conclude, from the available information, that in essence it has been shown that cellular damage in vitro was associated with lysosomal alterations before any ( 1) other pathologic alterations could be detected. Furthermore, BITENSKY used acid phosphatase reactions as a test for the fragility of lysosomes and their lipidprotein membrane. This author noted that a more intensive acid phosphatase is accompanied by an activation of the lysosomes and an increase in their permeability. With further damage of this organelle, diffusion of the enzymes into the cytoplasm occurs. The release of acid lytic enzymes will initiate lysis of the intracellular (13) constitutents which eventually may result in necrobiosis. Of particular interest is also the recent report of WEISSMAN & THOMAS (37)

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on steroids, lysosomes and systemic lupus erythematosus. These studies have disclosed in particular that activation by sunlight of lupus erythematosus in patients is expressed by the appearance of giant bullae with subepidermal detachment and some cellular necrosis, and there is a dramatic response of the related histopathology to adrenal glucocorticoids and an equally dramatic rebound following withdrawal of these agents. The protection by hydrocortisone of ultraviolet irradiation damage (36) of lysosomes in vivo and in vitro was also demonstrated. In relation to these findings it is important to mention that, 1) the inflammatory reactions described in collagenous diseases (to which lupus erythematosus belongs) and experimental allergic encephalomyelitis are considered of an hyperergic character and allergic in origin (11, 28), and 2) administration of cortisone to guinea pigs prior to injection of the encephalitogenic antigens (12, 17) has inhibited, in various degrees, the development of the clinical and histopathological reactions of the experimental allergic encephalomyelitis. Therefore, on the basis of the afore-mentioned electron microscope observations and because of the remarkable similarities between the protective effects of cortisone on lysosomes in vivo and in vitro (37, etc.), and of the tissue reactions in lupus erythematosus (37) and experimental allergic encephalomyelitis (12, 17), it seems suggestive that, at least in some phases a common pathogenic factor may exist in the development of these lesions and the allergic reactions. From the present preliminary study it appears to us that the lysosomes merit particular attention.

Conclusions I t is suggested in this report that the damage of the fine lysosomal structures and the release of lysosomal proteases as well as lysozymes may initiate a chain of reaction which, through tissue autolysis, could eventually lead to a process of necrobiosis, hyperergic inflammatory reactions and autosensitizations. These latter pathologic processes are considered as representative gradient phases of an iso-allergic mechanism. I n support of such a concept it should be considered that, a) the fine structural alterations of the lysosomes in the central nervous system of prenatally irradiated Macacus rhesus monkeys are similar to those which are associated with necrobiosis or autolysis in in vivo as well as in vitro experiments; b) similar pathological processes of lysosomal origin & THOMAS (37) in relation to systemhave recently been discussed by WEISSMAN ic lupus erythematosus; c) the inflammatory reaction of the irradiated neural tissue resembles the hyperergic type of inflammation and necrobiotic processes of the experimental allergic encephalomyelitis; and d ) the stability of the lysosoma1 fragility or permeability is protected and some of the tissue damage in lupus erythematosus and experimental allergic encephalomyelitis prevented in various degrees by preliminary administration of cortisone.

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Acknowledgements The technical assistance of Miss E. Forrester and Mr. S. Keoseian is gratefully acknowledged. These research investigations were supported in part by U.S. P. H. Grant Grant B-2 116 (C2) administered through Dr. R. Rugh.

SUMMARY This report is concerned principally with effects of roentgen irradiation upon the lysosomes of the fetal central nervous system of Macacus rhesus monkeys. Fundamental ultrastructural, cytochemical, functional and pathologic aspects of the lysosome organelle are reviewed in the light of present day knowledge, and various ultrastructural alterations of lysosomes as revealed by electron microscope investigations are described.

ZUSAMMENFASSUNG Dieser Bericht beschrankt sich grundsatzlich auf die Wirkungen der Rontgenstrahlen auf die Lysosome des ZNS von Macacus Rhesus Affen. Es wird nach dem Stand des heutigen Wissens eine ubersicht iiber grundsatzliche ultrastrukturelle, cytochemische, funktionelle und pathologische Aspekte gegeben und verschiedene ultrastrukturelle Veranderungen von Lysosomen, die elektronenmikroskopisch entdeckt wurden, beschrieben.

RESUME Ce travail est consacrt principalement aux effets de l’irradiation roentgen sur les lysosomes du systtme nerveux central dy foetus de singes Macacus rhesus. A la lumitre des connaissances actuelles, l’auteur rappelle les caractPres fondamentaux ultrastructurels, cytochimiques, fonctionnels et pathologiques des lysosomes et il dtcrit diverses altkrations ultrastructurelles des lysosomes montrkes par Ies examens au microscope Clectronique.

REFERENCES 1. BITENSKY L.: Cytotoxic action of antibodies. Brit. med. Bull. 19 (1963), 241.

2. - The reversible activation of lysosomes in normal cells and the effects of pathological 3. 4.

5.

6.

conditions. In: Lysosornes, Ciba Found. Symp., de Rueck, A.V.S. and Cameron M. P. editors, p. 362, Little, Brown and Co., Boston 1963. DAEMS W. TFI.: Mouse liver lysosomes and storage. A morphological and histochemical study. p. 89, Drukkerijh “Luctor et Emergo”, Leiden 1962. DE DUVEC.: From cytases to lysosomes. In: Lysosomes, Pathology Syniposium, Fed. Proc. 23 (1964), 1045. - A new group of cytoplasmic particles. In: Subcellular particles. Hayashi T., editor, p. 128. Ronald Press Co., New York 1959. - PRESSMAN B. C., GIANETTO R., WATTIAUX R. and APPELMANS F. : Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue. Biochem. J. 60 (1955), 6p4.

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7. DINGLE J. T.: Action of vitamin A on the stability of lysosomes in vivo and in vitro. In: Lysosomes, Ciba Found. Symp., de Rueck A. V. S. and Cameron M. P., editors, p. 384. Little, Brown & Co., Boston 1963. 8. DUMONDE D. C., BITENSKY L. and CHAYEN J.: Quoted by bibl. ref. 1. 9. - WALTERC. M., BITENSKY L., CUNNINGHAM G. Y. and CHAYEN J.: Intracellular response to an iso-immune reaction at the surface of ascites tumor cells. Nature 192 (1961), 1302. A. B.: Localization of acid phosphatase activity in hepatic 10. ESSNERE. and NOVIKOFF lysosomes by means of electron microscopy. J. biophys. biochem. Cytol. 9 (1961), 773. 11. FERRAROA. and ROIZINL. : Hyperergic encephalomyelitides following exanthematic diseases, infectious diseases and vaccination. J. Neuropath. exp. Neurol. 16 (1957), 423. 12. - - Experimental allergic encephalomyelitis during and following cortone acetate treatment. J. Neuropath. exp. Neurol. 12 (1953), 373. 13. GREENH., FLEISCHER R. A., BARROWP. and GOLDBERC B.: The cytotoxic action of immune gamma globulin and complement on Krebs ascites tumor cells. 11. Chemical studies. J. exp. Med. 109 (1959), 511. 14. HIRCH J. G. and COHNZ . A. : Digestive and autoIytic functions in phagocytic cells. In: Lysosomes. Pathology Symposium. Fed. Proc. 23 (1964), 1023. 15. HOLTS. J.: Factors governing the validity of staining methods for enzymes and their bearing upon the Gomori and phosphatase technique. Exp. Cell Res. 7 (1959), Suppl. 7. 16. - and HICKS R. M.: The localization of acid phosphatase in rat liver cells as revealed by combined cytochemical staining and electron microscopy. J. biophys. biochem. Cytol. 11 (1961), 47. 17. KABATE. A., WOLFA. and BEZERA. E.: Effect of cortisone on experimental acute disseminated encephalomyelitis. Trans. Amer. neurol. Ass. 76 (1951), 128. E. H., BROWNSON R. HH. and SUTERD. B.: Radiation-caused cytochemical chan18. KAGAN ges in neurons. Arch. Path. 74 (1962), 195. 19. KOENIG H. : Epileptogenic action of substances that interact with gangliosides. In vivo release of lysosomal enzymes as a disease mechanism. Trans. Amer. neurol. Ass. 87 (1962), 57. 20. Lysosomes, Pathology Symposium: Fed. Proc. 23 (1964), 1009. P. : Concerning the mechanism of the increased RNase activity 2 1. MAORD. and ALEXANDER in lymphatic cells following wholebody irradiation. Int. J. Radiat. Biol. 6 (1963), 93. 22. NOVIKOFFA. B. :Lysosomesand the physiology and pathology of cells. Biol. Bull. 1 17 ( 1959) 385. 23. - Biochemical and staining reactions of cytoplasmic constituents. In : Developing cell systems and their control, Rudnick I)., editor, p. 167. Ronald Press, New York 1960. 24. - Lysosomes and related particles. In: The Cell, Brachet J. and Mirsky A. E., editors, Vol. I1 p. 423. Academic Press, New York 1961. 25. - Lysosomes in the physiology and pathology of cells : contributions of staining methods. I n : Lysosomes, Ciba Found. Symp., de Rueck A. V. S. and Cameron M. P., editors, p. 36. Little, Brown & Co., Boston 1963. H. and DE DUVEC.: Electron microscopy of lysosome-rich fractions from 26. - BEAUFAY rat liver. J. biophys. biochem. Cytol. 179 (1956), Suppl. 2. 27. - ESSNER E. and QUINTANA N. : Golgi apparatus and lysosomes. In : Lysosomes. Pathology Symp. Fed. Proc. 23 (1964), 1010.

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ROIZINL. and KOLBL. C. : Considerations on the neuropathologic pleomorphism and histogenesis of the lesions of experimental allergic encephalomyelitis in non-human species. (Comparative morphologic and histochemical studies) In: Allergic Encephalomyelitis, Kies M. W. and Alvord E. C., editors, p. 5. Ch. C. Thomas Springfield 1959. - RUCHR. and KAUFMAN M.A.: Neuropathologic investigations of the x-irradiated embryo rat brain. J. Neuropath. exp. Neurol. 21 (1962), 219. - - - Effects of ionizing radiation on the rat embryo central nervous system at the cellular and ultracellular levels. The 2nd Internat. Symposium on : The response of nervous system to ionizing radiation. Little, Brown & Co., Boston 1964. ROTHJ. S., BUKOVSKY J. and EICHELH. J. : The effect of whole-body x-irradiation on the activity of some acid hydrolases in homogenates and subcellular fraction of rat spleen. Radiat. Res. 16 (1962) 27. TAPPEL A. L., SAWANT P. L. and SHIBKO S. : Lysosomes, distribution in animals, hydrolytic capacity and other properties. In: Lysosomes, Ciba Found. Symp., de Reuck A. V. S. and Cameron M. P., editors, p. 78, Little, Brown & Co., Boston 1963. J. L. : Concluding remarks. In: Lysosomes: Pathology Symposium. Fed. VANLANCKER Proc. 23 (1964), 1050. VANWACENEN G. and ASLINC C. M7.: Ossification in the fetal monkey (Macaca mulatta). Amer. J. Anat. 114 (1964), 107. G., BARLAND P. and WIEDERMANN G.: Role of lysosomes in streptolysin WEISSMANN S-induced arthritis. Clin. Res. Proc. 12 (1964), 240. - and FELLH. B.: Effect of hydrocortisone on the response of fetal rat skin in culture to ultraviolet irradiation. J. exp. Med. 116 (1962), 365. - and THOMAS L.: Steroids, lysosomes and systemic lupus erythematosus. Bull. N. Y. Acad. Med. 38 (1962), 779.