Exp Toxic Pathol 1993; 45: 55-59

Gustav Fischer Verlag Jena

Karolinska Institute, Stockholm, Sweden

Tubuloreticular structures in pyridoxine deficiency

A. G. S. DATSIS

With 2 figures

Received: November 26, 1991; Accepted: December 12, 1991

Address for correspondence: Dr. A. G. S. DATSIS, 31 Aristotelus street, 54631 Thessaloniki, Greece.

Key words: Tubulo-reticular structures; Undulating tubeles; Ergastroplasm; Crystalline inclusions; Endothelium; Pyridoxine deficiency; Kidney; Glomerulus

Summary

Electron microscopic studies on renal tissue obtained from animals subjected to pyridoxine deficiency have revealed the presence of round and/or tubular bodies in the cytoplasm of endothelial cells of the glomeruli. The bodies generally occur as aggregates in association with the endoplasmic reticulum. Along with the round forms other profiles are also observed. It is believed that they represent one of the many images of a system of undulating tubules.

Since these bodies occur irrespective of the presence of virus particles in the tissue under study it is concluded that they are not viral in nature.

Introduction

Numerous reports on ultrastructural observations have appeared in the literature concerning distinctive tubuloreticu­lar structures 18 to 26nm in diameter within the endoplasmic reticulum of cells of normal human and monkey tissues, in a wide variety of renal diseases, in several neoplasms and in a number of viral infections both in vivo and in vitro; details of these observations have been reviewed by the author (DATSIS 1972, 1973). The morphologic appearance of the particles has prompted their being considered viral or viruslike in nature, provoking speculation concerning the viral origin of the associated illnesses. However, the finding of these tubuloreticular structures in a wide spectrum of pathological conditions, their most frequent restriction to endothelial cells, and their nearly constant occurrence within the endo­plasmic reticulum suggest that they may repesent a host-cell response rather than a virus or virus-like material.

The purpose of the present communication is to report the occurrence of endothelial tubuloreticular inclusions in kid­ney tissue obtained from animals subjected to vitamin B6 (pyridoxine) deficiency. Furthermore, it is aiming at present­ing new electron microscopic observations demonstrating not merely that the tubuloreticular structures are contained within elements of the endoplasmic reticulum, but that they

do arise from it by a process of invagination or budding. These findings suggest that the tubuloreticular structures are, indeed, an intimate part of or are derived from the endoplas­mic reticulum and are thus absolutely not likely to represent virus particles.

Materials and methods

Adult male Sprague-Dawley albino rats, ranging in weight from 150 to 200g were used in this investigation. The animals were fed a high protein diet (40 % casein) devoid of pyridoxine to which 100 mg of desoxypyridoxine were added per 100 g of food. Operative and histological techniques employed in this study will be reported elsewhere (DATSIS 1991).

Results

Cytoplasmic inclusions similar to those reported occuring in a variety of disease conditions, as discussed in the next section, were found in the endothelial cells of the glomerular capillaries (figures 1 and 2). The endothelial cells contain aggregates of small round bodies, some of which appear to be "hollow". The aggregates are delimited by a well-defined smooth membrane which at many points display an apparent connection with elements of the smooth and rough surfaced endoplasmic reticulum. The round bodies frequently pro­duce crystalline patterns (figure 2). At higher magnifications some of these structures display an appearance of round, smooth-membrane-bound structures with electron - lucent "cores" (figure 2, Inset). Profiles of co-existent bodies, however, indicate that they may not be spherical but rather tubular. Examination of the electron micrographs suggests that the apparent crystalline pattern of granules is the result of a transverse plane of section passing through a system of undulating anastomosing tubules. Both tubules and granules measured approximately 25 nm in diameter. The undulating

Exp Toxic Pathol 45 (1993) 1 55

Exp Toxic Pathol 1993; 45: 55-59

Gustav Fischer Verlag Jena

Karolinska Institute, Stockholm, Sweden

Tubuloreticular structures in pyridoxine deficiency

A. G. S. DATSIS

With 2 figures

Received: November 26, 1991; Accepted: December 12, 1991

Address for correspondence: Dr. A. G. S. DATSIS, 31 Aristotelus street, 54631 Thessaloniki, Greece.

Key words: Tubulo-reticular structures; Undulating tubeles; Ergastroplasm; Crystalline inclusions; Endothelium; Pyridoxine deficiency; Kidney; Glomerulus

Summary

Electron microscopic studies on renal tissue obtained from animals subjected to pyridoxine deficiency have revealed the presence of round and/or tubular bodies in the cytoplasm of endothelial cells of the glomeruli. The bodies generally occur as aggregates in association with the endoplasmic reticulum. Along with the round forms other profiles are also observed. It is believed that they represent one of the many images of a system of undulating tubules.

Since these bodies occur irrespective of the presence of virus particles in the tissue under study it is concluded that they are not viral in nature.

Introduction

Numerous reports on ultrastructural observations have appeared in the literature concerning distinctive tubuloreticu­lar structures 18 to 26nm in diameter within the endoplasmic reticulum of cells of normal human and monkey tissues, in a wide variety of renal diseases, in several neoplasms and in a number of viral infections both in vivo and in vitro; details of these observations have been reviewed by the author (DATSIS 1972, 1973). The morphologic appearance of the particles has prompted their being considered viral or viruslike in nature, provoking speculation concerning the viral origin of the associated illnesses. However, the finding of these tubuloreticular structures in a wide spectrum of pathological conditions, their most frequent restriction to endothelial cells, and their nearly constant occurrence within the endo­plasmic reticulum suggest that they may repesent a host-cell response rather than a virus or virus-like material.

The purpose of the present communication is to report the occurrence of endothelial tubuloreticular inclusions in kid­ney tissue obtained from animals subjected to vitamin B6 (pyridoxine) deficiency. Furthermore, it is aiming at present­ing new electron microscopic observations demonstrating not merely that the tubuloreticular structures are contained within elements of the endoplasmic reticulum, but that they

do arise from it by a process of invagination or budding. These findings suggest that the tubuloreticular structures are, indeed, an intimate part of or are derived from the endoplas­mic reticulum and are thus absolutely not likely to represent virus particles.

Materials and methods

Adult male Sprague-Dawley albino rats, ranging in weight from 150 to 200g were used in this investigation. The animals were fed a high protein diet (40 % casein) devoid of pyridoxine to which 100 mg of desoxypyridoxine were added per 100 g of food. Operative and histological techniques employed in this study will be reported elsewhere (DATSIS 1991).

Results

Cytoplasmic inclusions similar to those reported occuring in a variety of disease conditions, as discussed in the next section, were found in the endothelial cells of the glomerular capillaries (figures 1 and 2). The endothelial cells contain aggregates of small round bodies, some of which appear to be "hollow". The aggregates are delimited by a well-defined smooth membrane which at many points display an apparent connection with elements of the smooth and rough surfaced endoplasmic reticulum. The round bodies frequently pro­duce crystalline patterns (figure 2). At higher magnifications some of these structures display an appearance of round, smooth-membrane-bound structures with electron - lucent "cores" (figure 2, Inset). Profiles of co-existent bodies, however, indicate that they may not be spherical but rather tubular. Examination of the electron micrographs suggests that the apparent crystalline pattern of granules is the result of a transverse plane of section passing through a system of undulating anastomosing tubules. Both tubules and granules measured approximately 25 nm in diameter. The undulating

Exp Toxic Pathol 45 (1993) 1 55

nature of the said tubules was most clearly depicted in fortuitous longitudinal sections (figure 2, Inset). Membran­ous elements of the smooth-surfaced type endoplasmic reticulum appeared to be continuously connected with the arrays of the tubular structures. In some instances, the plane of section was such as to produce images of greater complexity. Furthermore, circular profiles delimited by two membranes were also encountered. A careful examination strongly suggested that the latter were formed by two

56 Exp Toxic Pathol 45 (1993) I

Fig. 1. Glomerulus from the kidney of a rat subjected to vitamin B6 (pyridox­ine) deficiency for 16 days. Focal glomerular sclerosis with a marked thickening of the capillary walls is noticed. Fixation in 1 %-s-collidine buf­fered OS04, stained with Toluidine Blue, x250.

Fig. 2. Electron micrograph illustrat­ing portion of the glomerulus depicted in fig. 1. There appears to be a wide­spread thickening of the capillary base­ment membrane (bm), while the en­dothelial layer covering the membrane is markedly swollen (end). The en­dothelium contains aggregates arranged in a crystalline pattern; a single, smooth membrane, displaying points of app~ent continuity with the ergasto­plasmic membranes is seen delimiting the endothelial inclusions. Fixation in I %-s-collidine buffered OS04, stained with uranyl acetate and lead hydroxide. X22,600. Inset: High magnification of a section through a portion of the endothelial in­clusion showing the tubular nature of the subunits of the complex. Round profiles formed by undulating tubules, and displaying "hollow cores" are quite prominent. Elements of the endoplas­mic reticulum appear to be continuous­ly connected with the array of the tubu­lar structure (arrows). Fixation as in fig. 2, X32,490.

undulating tubules lying within the thickness of the section with the crest of one lying opposite the trough of the other.

Discussion

Tubuloreti<;ular structures similar to the ones observed in the present investigation have been observed in different cells of normal tissues and in cells of humans with various

nature of the said tubules was most clearly depicted in fortuitous longitudinal sections (figure 2, Inset). Membran­ous elements of the smooth-surfaced type endoplasmic reticulum appeared to be continuously connected with the arrays of the tubular structures. In some instances, the plane of section was such as to produce images of greater complexity. Furthermore, circular profiles delimited by two membranes were also encountered. A careful examination strongly suggested that the latter were formed by two

56 Exp Toxic Pathol 45 (1993) I

Fig. 1. Glomerulus from the kidney of a rat subjected to vitamin B6 (pyridox­ine) deficiency for 16 days. Focal glomerular sclerosis with a marked thickening of the capillary walls is noticed. Fixation in 1 %-s-collidine buf­fered OS04, stained with Toluidine Blue, x250.

Fig. 2. Electron micrograph illustrat­ing portion of the glomerulus depicted in fig. 1. There appears to be a wide­spread thickening of the capillary base­ment membrane (bm), while the en­dothelial layer covering the membrane is markedly swollen (end). The en­dothelium contains aggregates arranged in a crystalline pattern; a single, smooth membrane, displaying points of app~ent continuity with the ergasto­plasmic membranes is seen delimiting the endothelial inclusions. Fixation in I %-s-collidine buffered OS04, stained with uranyl acetate and lead hydroxide. X22,600. Inset: High magnification of a section through a portion of the endothelial in­clusion showing the tubular nature of the subunits of the complex. Round profiles formed by undulating tubules, and displaying "hollow cores" are quite prominent. Elements of the endoplas­mic reticulum appear to be continuous­ly connected with the array of the tubu­lar structure (arrows). Fixation as in fig. 2, X32,490.

undulating tubules lying within the thickness of the section with the crest of one lying opposite the trough of the other.

Discussion

Tubuloreti<;ular structures similar to the ones observed in the present investigation have been observed in different cells of normal tissues and in cells of humans with various

pathological disorders. A variety of different names have been attributed to these elements, such as "undulating tubu­les" (BASSOT 1966; CHANDRA 1968), "crystalline aggregates" (BUCCIARELLI et al. 1967), "crystal-lattice structures" (KIM and BOATMAN 1967), "tubular network" (VAN LENNEP and LANZIG 1967), "crystaline inclusions" (SEBUWUFU 1968), "cytoplasmic crystaline structures" (HURD et al. 1969), "filamentous tubular structures" (SINK­OVICS et al. 1969), "tubular inclusions" (BLOODWORTH and SHELP 1970), "crystalloids" (ESTES and CHEVILLE 1970), "tubular arrays" (DATSIS 1972), "tubuloreticular structures" (SCHAFF et al. 1972; DATSIS 1973). The last term appears describing more precisely the appearance of the structures in reference without limiting their variable morphological aspects which may, indeed, range from loosely interwoven tubules to extremely orderly patterns of paracrystaline con­formations. Since the structures have been found not exclu­sively inside the cytoplasm in close association with the endoplasmic reticulum, but also in the perinuclear space and even inside the nucleus (GYORKEY et al. 1969), no reference to their localization inside the cell is made in the last term mentioned above.

The role of these structures in the cell is still obscure and a matter of speculation. The different conditions under which they have been found have been reviewed by SCHAFF et al. (1972) and DATSIS (1972, 1973). Under certain circumstan­ces they may represent a normal component of cells being in highly specialized metabolic functions as in the dendritic organ of the catfish (VAN LENNEP and LEN ZIG 1967), and in the photogenic cells of the polynoid worm Elytra (BASSOT 1966). Better known, however, is the frequency with which such structures are found in various cells in certain autoim­mune diseases and other renal diseases. To this end, most frequently they have been observed in glomerular endothe­lial cells in systemic lupus erythematosus (GYORKEY and SINKOVICS 1969; HURD et al. 1969; KAWANO et al. 1969; PINCUS et al. 1970; SINKOVICS et al. 1969; DE MARTINO et al. 1969; GRAUSZ et al. 1970; HAAS and YUNIS 1970; DATSIS 1991), and they have been associated with endothe­lial cells of different organs in a number of autoimmune diseases (NORTON et al. 1970; SHEARN et al. 1970). The structures have also been seen in a variety of tumors (GYOR­KEY et al. 1969; LOMBARD et al. 1967; MUNROE et al. 1964). and in virus-infected cells either in vivo (BUCCIARELLI et al. 1967; ESTES and CHEVILLE 1970), or in vitro (D A VID­FERREIRA and MANAKER 1965; CHANDRA, 1968).

From the preceding it is apparent that we are confronted with a major problem in elucidating a common denominator for the presence of these structures. The most impressing factor thus far is their preference for cells belonging to the reticuloendothelial system. However, an evaluation of their meaning, even in these cells seems to be difficult since they have been observed in different cellular sites, viz., the nucleus and the cytoplasm. Due to their most frequent presence in cells of the reticuloendothelial system, it was proposed that the tubuloreticular structures in reference reflect a response of these cells to pathological conditions, thus possibly representing a marker for cellular injury. Their

possible involvement in the production of immunoglobulins should also be considered (DATSIS 1991). Although it is difficult to establish the frequency of these structures in normal tissues, most studies indicate that the aggregates are found most often in disease states. The morphologic charac­ter of the aggregates has differed in the variety of circum­stances in which they have been found. In normal tissues, neoplastic conditions and virus-infected tissues they have usually appeared as a rather tightly packed array of particles that have only occasionally been recognized as tubular in nature (CHANDRA, 1968). In contrast, in most ofthe material from patients with lupus nephritis and other renal diseases, the appearance is that of loosely arrayed tubules. A number of features, however, are common to both types of arrays: (a) their occurrence related to and most often within the elements of the endoplasmic reticulum, and (b) their localization in endothelial cells or less frequently in macrophages. These common features have suggested that the several observations may all concern the same structures, and that variations in appearance of the structures may well be related to the packing density of the aggregates.

Great strides have been made in unifying the various morphologic types of tubular or crystaline in pattern arrays found within the cells in the pathological conditions men­tioned above, on the basis of their intimate relationship to viral infections. But to limit consideration of these apparent tubular structures to the general concept of intracellular viral inclusions, simply because they have been documented in diseases of a viral origin, would not be the most constructive approach. There would then be a tendency to dismiss further investigation of the biological importance of this cytoplas­mic reactivity. While there are apparent points of similarity between these tubular and/or crystalline arrays and the viral particles, to permit this general conceptualization deter specific consideration of these intracellular tubular structures would only obstruct rather than clarify the basic cytoplasmic tubuloreticular nature of this cytoplasmic and/or nuclear change.

The several reports concerning the appearance of apparent tubular structures in the kidneys of patients with clinically proven systemic lupus erythematosus have generated much interest in a possible viral etiology, since these particles do bear some resemblance to myxoviruses. Although several investigators have not found similar particles in normal renal tissues, the particles have persistently been found in biopsies from patients with lupus nephritis and other renal diseases (BATTIFORA and MARKOWITZ 1969; DUFFY, 1969; DE MAR­TINO et al. 1969; GRAUSZ et al. 1970; GYORKEY et al. 1969; NORTON 1969; HURD et al. 1969; KAWANO et al. 1969; SINKOVICE et al. 1969; PINCUS et al. 1970; HAAS and YUNIS 1970; DA TSIS 1972, 1973, 1991). The particles size of 20 to 25nm given in most of the reports is, however, larger than the nucleocapsid of the myxovirus or paramyxovirus group (DAVID et al. 1967; ROIZMAN 1971).

Data obtained in the present investigation indicate the direct relation of these tubuloreticular structures to the endo­plasmic reticulum, as physical connections between these two sub-cellular structures have been documented. This,

Exp Toxic Pathol 45 (1993) 1 57

pathological disorders. A variety of different names have been attributed to these elements, such as "undulating tubu­les" (BASSOT 1966; CHANDRA 1968), "crystalline aggregates" (BUCCIARELLI et al. 1967), "crystal-lattice structures" (KIM and BOATMAN 1967), "tubular network" (VAN LENNEP and LANZIG 1967), "crystaline inclusions" (SEBUWUFU 1968), "cytoplasmic crystaline structures" (HURD et al. 1969), "filamentous tubular structures" (SINK­OVICS et al. 1969), "tubular inclusions" (BLOODWORTH and SHELP 1970), "crystalloids" (ESTES and CHEVILLE 1970), "tubular arrays" (DATSIS 1972), "tubuloreticular structures" (SCHAFF et al. 1972; DATSIS 1973). The last term appears describing more precisely the appearance of the structures in reference without limiting their variable morphological aspects which may, indeed, range from loosely interwoven tubules to extremely orderly patterns of paracrystaline con­formations. Since the structures have been found not exclu­sively inside the cytoplasm in close association with the endoplasmic reticulum, but also in the perinuclear space and even inside the nucleus (GYORKEY et al. 1969), no reference to their localization inside the cell is made in the last term mentioned above.

The role of these structures in the cell is still obscure and a matter of speculation. The different conditions under which they have been found have been reviewed by SCHAFF et al. (1972) and DATSIS (1972, 1973). Under certain circumstan­ces they may represent a normal component of cells being in highly specialized metabolic functions as in the dendritic organ of the catfish (VAN LENNEP and LEN ZIG 1967), and in the photogenic cells of the polynoid worm Elytra (BASSOT 1966). Better known, however, is the frequency with which such structures are found in various cells in certain autoim­mune diseases and other renal diseases. To this end, most frequently they have been observed in glomerular endothe­lial cells in systemic lupus erythematosus (GYORKEY and SINKOVICS 1969; HURD et al. 1969; KAWANO et al. 1969; PINCUS et al. 1970; SINKOVICS et al. 1969; DE MARTINO et al. 1969; GRAUSZ et al. 1970; HAAS and YUNIS 1970; DATSIS 1991), and they have been associated with endothe­lial cells of different organs in a number of autoimmune diseases (NORTON et al. 1970; SHEARN et al. 1970). The structures have also been seen in a variety of tumors (GYOR­KEY et al. 1969; LOMBARD et al. 1967; MUNROE et al. 1964). and in virus-infected cells either in vivo (BUCCIARELLI et al. 1967; ESTES and CHEVILLE 1970), or in vitro (D A VID­FERREIRA and MANAKER 1965; CHANDRA, 1968).

From the preceding it is apparent that we are confronted with a major problem in elucidating a common denominator for the presence of these structures. The most impressing factor thus far is their preference for cells belonging to the reticuloendothelial system. However, an evaluation of their meaning, even in these cells seems to be difficult since they have been observed in different cellular sites, viz., the nucleus and the cytoplasm. Due to their most frequent presence in cells of the reticuloendothelial system, it was proposed that the tubuloreticular structures in reference reflect a response of these cells to pathological conditions, thus possibly representing a marker for cellular injury. Their

possible involvement in the production of immunoglobulins should also be considered (DATSIS 1991). Although it is difficult to establish the frequency of these structures in normal tissues, most studies indicate that the aggregates are found most often in disease states. The morphologic charac­ter of the aggregates has differed in the variety of circum­stances in which they have been found. In normal tissues, neoplastic conditions and virus-infected tissues they have usually appeared as a rather tightly packed array of particles that have only occasionally been recognized as tubular in nature (CHANDRA, 1968). In contrast, in most ofthe material from patients with lupus nephritis and other renal diseases, the appearance is that of loosely arrayed tubules. A number of features, however, are common to both types of arrays: (a) their occurrence related to and most often within the elements of the endoplasmic reticulum, and (b) their localization in endothelial cells or less frequently in macrophages. These common features have suggested that the several observations may all concern the same structures, and that variations in appearance of the structures may well be related to the packing density of the aggregates.

Great strides have been made in unifying the various morphologic types of tubular or crystaline in pattern arrays found within the cells in the pathological conditions men­tioned above, on the basis of their intimate relationship to viral infections. But to limit consideration of these apparent tubular structures to the general concept of intracellular viral inclusions, simply because they have been documented in diseases of a viral origin, would not be the most constructive approach. There would then be a tendency to dismiss further investigation of the biological importance of this cytoplas­mic reactivity. While there are apparent points of similarity between these tubular and/or crystalline arrays and the viral particles, to permit this general conceptualization deter specific consideration of these intracellular tubular structures would only obstruct rather than clarify the basic cytoplasmic tubuloreticular nature of this cytoplasmic and/or nuclear change.

The several reports concerning the appearance of apparent tubular structures in the kidneys of patients with clinically proven systemic lupus erythematosus have generated much interest in a possible viral etiology, since these particles do bear some resemblance to myxoviruses. Although several investigators have not found similar particles in normal renal tissues, the particles have persistently been found in biopsies from patients with lupus nephritis and other renal diseases (BATTIFORA and MARKOWITZ 1969; DUFFY, 1969; DE MAR­TINO et al. 1969; GRAUSZ et al. 1970; GYORKEY et al. 1969; NORTON 1969; HURD et al. 1969; KAWANO et al. 1969; SINKOVICE et al. 1969; PINCUS et al. 1970; HAAS and YUNIS 1970; DA TSIS 1972, 1973, 1991). The particles size of 20 to 25nm given in most of the reports is, however, larger than the nucleocapsid of the myxovirus or paramyxovirus group (DAVID et al. 1967; ROIZMAN 1971).

Data obtained in the present investigation indicate the direct relation of these tubuloreticular structures to the endo­plasmic reticulum, as physical connections between these two sub-cellular structures have been documented. This,

Exp Toxic Pathol 45 (1993) 1 57

under no circumstances has been a feature of myxovirus or of paramyxovirus infections in which the virus is found within the nuclei or free in the cellular cytoplasm (ROIZMAN 1971).

The biological existence of systems of "undulating tubu­les" having organized or disorganized orientations were reported by CHANDRA (1968). In that study substantial proof for the presence of mutually perpendicular systems of undulating tubles forming a sign of "plus" was the most often encountered. Since many other orientations of the tubules are possible, various images can often be observed in submicroscopic studies. It is apparent that the most fre­quently observed images would be the round bodies or profiles. Aggregates of round profiles were observed in tumors induced by Rous sarcoma virus (BUCCIARELLI et al. 1967; MUNROE et al. 1964), in the Sticker sarcoma of the dog (LOMBARD et al. 1967) and in cell cultures infected with rubella virus (KIM and BOA TMANN 1967). The evidence indicates that they represent images of undulating micro­tubules by certain planes of section. In an organized state, the tubules could produce images exhibiting a crystalline or tubular pattern. The round bodies cannot, therefore, be conceived as viral in nature.

To date, the morphogenesis of the tubuloreticular struc­tures is not known. In one report it was proposed that these structures may originate directly from the endoplasmic reticulum (CHANDRA 1968; BASSOT 1966), while in another it was hypothesized that they may be formed by the consens­ing of granular material found in circumscribed areas of the dilated endoplasmic reticulum or that they even appear as the result of the release of ribosomes from parts of the rough­surfaced endoplasmic reticulum (FILSHIE et al. 1969). In this report, the development of a rod-shaped virus was studied in midgut cells of an adult insect. It was found that the tubuloreticular structures appear in the cytoplasm at a time when viral DNA synthesis starts in the nucleus. Later on, the presence of high amounts of thymidine in the nucleus was paralleled by the enlargement of the tubuloreticular struc­tures. In the case studied, virus particles mature inside the nucleus. Hence, it is apparent that the tubuloreticular struc­tures probably are the manifestation of a secondary effect on the cells related to the virus infection, but the structures themselves do not represent viral precursors per se.

Observations by SCHAFF et al. (1972), clearly show that the tubuloreticular structures are in close contact with the rough-surfaced endoplasmic reticulum. Nevertheless, they are presumably of a somewhat different chemical composi­tion, since their behaviour towards digestive enzymes, espe­cially pronase, differs from that of the membranes of the reticulum. Cytochemical studies by the same group provided substantial evidence for the proteinaceous nature of these structures. The presence of nucleic acids in the tubuloreticu­lar structures appears to be all unlikely, since the staining and digestion methods utilized in that study to characterize such substances failed to give positive results. Thus with regard to the biochemical nature of these tubuloreticular structures, it could be concluded that these structures are predominantly made up of proteins, especially those of the

58 Exp Toxic Pathol45 (1993) 1

acidic type, since they are most easily attacked by papain (SCHAFF et al. 1972; DATSIS 1991 a). It could also be deduced that viral proteins should not be considered to be a component of the tubuloreticular structures in reference.

On the basis of the aforementioned theories and observa­tions, it is apparent that the origin as well as the significance of these structures is still a matter of dispute, but considering the data available thus far, the author would like to support those previously published views in which the structures under discussion have been considered to represent a cellular reaction to factors mostly unknown today. Further investiga­tions are necessary to elucidate their specific role in the cell.

References

1. BASSOT JM: Une forme microtubulaire et paracristalline de reticulum endoplasmique dans les photocytes des Annelides Polynoinae. J Cell BioI 1966; 31: 135-158.

2. BATTIFORA HA, MARKOWITZ AS: Nephrotoxic nephritis in monkeys: sequential light, immunofluorescence and elec­tron microscopic studies. Am J Patho11969; 55: 267-281.

3. BUCCIARELLI E, RABOTTI GF, DALTON AJ: Ultrastructure of meningeal tumors in dogs with Rous sarcoma virus. J Natl Cancer Inst 1967; 38: 359-381.

4. CHANDRA S: Undulating tubules associated with endoplas­mic reticulum in pathological tissues. Lab Invest 1968; 18: 422-428 (1968).

5. DATSIS AGS: Cytoplasmic tubular Arrays in Latent Chronic Glomerulonephritis. Virchows Arch Abt A Path Anat 1972 357: 187-197.

6. DATSIS AGS: Endothelial Inclusions in Congenital Infantile Nephrosis. Virchows Arch Abt A Path Anat 1973; 359: 105-109.

7. DATSIS AGS: Pathogenesis of Lupus Nephritis. An Interpretation of the Ultrastructural Lesion. (In manuscript, 1991).

8. DATSIS AGS: Cytochemical studies on Endothelial tubuloreticular structures (In manuscript, 1991).

9. DAVID-FERREIRA JF, MANAKER RA: An electron micro­scopic study of the development of a mouse hepatitis virus in tissue culture cells. J Cell BioI 1965; 24: 57-65.

10. DAVIS BD, DULBECCO R, EISEN HN: Microbiology: A Text emphasizing molecular and genetic aspects of microbiology and immunology, and the relations of bacteria, fungi and viruses to human disease. New York. Harper & Row, 1967, p. 1313.

11. DE MARTINO C, ACCINIC L, ANDRES GB, et al.: Tubular structures associated with the endoplasmic reticulum in glomerular capillaries of rhesus monkey and nephritic man. Z Zellforsch Mikrosk Anat 1969; 97: 502-511

12. DUFFY JL: Myxovirus-like particles in lipoid nephrosis. N Engl J Med 169: 281: 562-563.

13. ESTES PC, CHEVILLE NF: The ultrastructure of vascular lesions in equine viral arteritis. Am J Pathol 1970; 58: 235-253.

14. FILS HIE BK, REHACEK J: Studies of the morphology of Murray Valley encephalitis and Japanese encephalitis vir­uses growing in cultured mosquito cells. Virology 1968; 34: 435-443.

15. GRAUSZ H, EARLY LE, STEPHENS BG: Diagnostic import of virus-like particles in the glomerular epithelium of

under no circumstances has been a feature of myxovirus or of paramyxovirus infections in which the virus is found within the nuclei or free in the cellular cytoplasm (ROIZMAN 1971).

The biological existence of systems of "undulating tubu­les" having organized or disorganized orientations were reported by CHANDRA (1968). In that study substantial proof for the presence of mutually perpendicular systems of undulating tubles forming a sign of "plus" was the most often encountered. Since many other orientations of the tubules are possible, various images can often be observed in submicroscopic studies. It is apparent that the most fre­quently observed images would be the round bodies or profiles. Aggregates of round profiles were observed in tumors induced by Rous sarcoma virus (BUCCIARELLI et al. 1967; MUNROE et al. 1964), in the Sticker sarcoma of the dog (LOMBARD et al. 1967) and in cell cultures infected with rubella virus (KIM and BOA TMANN 1967). The evidence indicates that they represent images of undulating micro­tubules by certain planes of section. In an organized state, the tubules could produce images exhibiting a crystalline or tubular pattern. The round bodies cannot, therefore, be conceived as viral in nature.

To date, the morphogenesis of the tubuloreticular struc­tures is not known. In one report it was proposed that these structures may originate directly from the endoplasmic reticulum (CHANDRA 1968; BASSOT 1966), while in another it was hypothesized that they may be formed by the consens­ing of granular material found in circumscribed areas of the dilated endoplasmic reticulum or that they even appear as the result of the release of ribosomes from parts of the rough­surfaced endoplasmic reticulum (FILSHIE et al. 1969). In this report, the development of a rod-shaped virus was studied in midgut cells of an adult insect. It was found that the tubuloreticular structures appear in the cytoplasm at a time when viral DNA synthesis starts in the nucleus. Later on, the presence of high amounts of thymidine in the nucleus was paralleled by the enlargement of the tubuloreticular struc­tures. In the case studied, virus particles mature inside the nucleus. Hence, it is apparent that the tubuloreticular struc­tures probably are the manifestation of a secondary effect on the cells related to the virus infection, but the structures themselves do not represent viral precursors per se.

Observations by SCHAFF et al. (1972), clearly show that the tubuloreticular structures are in close contact with the rough-surfaced endoplasmic reticulum. Nevertheless, they are presumably of a somewhat different chemical composi­tion, since their behaviour towards digestive enzymes, espe­cially pronase, differs from that of the membranes of the reticulum. Cytochemical studies by the same group provided substantial evidence for the proteinaceous nature of these structures. The presence of nucleic acids in the tubuloreticu­lar structures appears to be all unlikely, since the staining and digestion methods utilized in that study to characterize such substances failed to give positive results. Thus with regard to the biochemical nature of these tubuloreticular structures, it could be concluded that these structures are predominantly made up of proteins, especially those of the

58 Exp Toxic Pathol45 (1993) 1

acidic type, since they are most easily attacked by papain (SCHAFF et al. 1972; DATSIS 1991 a). It could also be deduced that viral proteins should not be considered to be a component of the tubuloreticular structures in reference.

On the basis of the aforementioned theories and observa­tions, it is apparent that the origin as well as the significance of these structures is still a matter of dispute, but considering the data available thus far, the author would like to support those previously published views in which the structures under discussion have been considered to represent a cellular reaction to factors mostly unknown today. Further investiga­tions are necessary to elucidate their specific role in the cell.

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patients with Systemic Lupus Erythematosus. N Engl J Med 1970; 283: 506-511 (1970).

16. GYORKEY F, MIN KW , SINKOVICS JG , et al. : Systemic Lupus Erythematol'us and myxovirus. N Engl J Med 1969; 280: 333-340 (1969) .

17. HAAS JE, YUNIS EJ: Tubular Inclusions of Systemic Lupus Erythematosus: Ultrastructural observations regarding their possible viral nature. Exp Mol Pathol 1970; 12: 257-263 .

18. HAAS JE, YUNIS EJ: Viral crystalline arrays in human Coxsackie myocarditis. Lab Invest 1970; 23: 442-446 (1970).

19. HURD ER, EIGENBRODT E, ZIFF M, et al.: Cytoplasmic tubular structures in kidney biopsies in Systemic Lupus Erythematosus. Arthritis Rheum 1969; 12: 541-542.

20. KAWANO K, MILLER L, KiMMESTIEL P : Virus-like struc­tures in Lupus Erythematosus. N Engl J Med 1969 ; 281: 1228-1229.

21. KIM KSW, BOATMAN ES: Electron microscopy of monkey kidney cell cultures infected with rubella virus. J Virol 1967 ; 1: 205-214.

22. LOMBARD C, CABANIE P , IZARD J : Images evoquant J'as­pect de virus dans les cellules du sarcome du Sticker. Microscopie 1967; 6 : 81 - 84.

23 . MUNROE JS , SHIPKEY F, ERLANDSON RA: Tumors induced in juvenile and adult primates by chicken sarcoma virus . Natl Cancer Inst Monogr 1964; 17: 365- 390.

24. NORTON WL: Endothelial inclusions in active lesions of Systemic Lupus Erythematosus . J Lab Clin Med 1969; 74: 369-379.

25 . PINCUS T, BLACKLOW NR, GRIMLEY PM: Glomerular microtubules of Systemic Lupus Erythematosus. Lancet 1970; 2: 1058-1061.

26. ROIZMAN, B.: Personal Communication (1972). 27. SEBUWUFU PH: Crystalline inclusions in normal primate

thymoblasts . Nature 1968 ; 218: 980-981. 28 . SHEARN MA, Tu WH, STEPHENS BG, et al. : Virus like

structures in Sjogrens syndrome. Lancet 1970; 1: 568-569.

29. SINKOVICS JG, GYORKEY F, THOMA GW: A rapidly fatal case of Systemic Lupus Erythematosus: structures resembl­ing viral nucleoprotein strands in the kidney and activities of lymphocytes in culture. Tex Rep BioI Med 1969; 27: 887-908.

30. VAN LENNEP EW, LANZIG WJR: The ultrastructure of glandular cells in the external dendritic organ of some marine catfish. J Ultrastruct Res 1967 ; 18: 333-350.

Exp Toxic Pathol 45 (1993) 1 59